Novel therapeutic targets in cancer

ABSTRACT

The present invention relates to novel sequences for use in detection, diagnosis and treatment of cancers, especially lymphomas. The invention provides cancer associated (CA) polynucleotide sequences whose expression is associated with cancer. The present invention provides CA polypeptides associated with cancer that are present on the cell surface and present novel therapeutic targets against cancer. The present invention further provides diagnostic compositions and methods for the detection of cancer. The present invention provides monoclonal and polyclonal antibodies specific for the CA polypeptides. The present invention also provides diagnostic tools and therapeutic compositions and methods for screening, prevention and treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to the following U.S. Applications: U.S.Ser. No. 10/034,650, filed Dec. 20, 2001; U.S. Ser. No. 10/035,832,filed Dec. 26, 2001; U.S. Ser. No. 10/004,113, filed Oct. 23, 2001; U.S.Ser. No. 09/997,722, filed Nov. 30, 2001; U.S. Ser. No. 10/085,117,filed Feb. 27, 2002; U.S. Ser. No. 10/087,192, filed Mar. 1, 2002; U.S.Ser. No. 10/322,281, filed Dec. 17, 2002; U.S. Ser. No. 10/322,696,filed Dec. 17, 2002, U.S. Ser. No. 10/331,053, filed Dec. 26, 2002; U.S.Ser. No. 10/330,773, filed Dec. 27, 2002; U.S. Ser. No. 10/367,094,filed Feb. 14, 2003; U.S. Ser. No. 10/388,838, filed Mar. 14, 2003, U.S.Ser. No. 10/417,375 filed Apr. 15, 2003, U.S. Ser. No. 10/461,862, filedJun. 13, 2003, U.S. Ser. No. 10/663,431 filed Sep. 15, 2003, U.S. Ser.No. 10/669,920 filed Sep. 23, 2003, U.S. Ser. No. 10/670,914 filed Sep.24, 2003, U.S. Ser. No. 10/674,575, filed Sep. 29, 2003, U.S. Ser. No.10/676,684 filed Sep. 30, 2003, and U.S. Ser. No. 10/692,382 filed Oct.22, 2003, all of which are expressly incorporated herein by reference intheir entirety.

DESCRIPTION OF ACCOMPANYING CD-ROMs

Tables 1-16 are filed herewith in CD-ROM in accordance with 37 C.F.R. §§1.52 and 1.58. Two identical copies (marked “Copy 1” and “Copy 2”) ofthis CD-ROM are submitted.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090214546A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Contents of the CD-ROM disks submitted herewith are hereby incorporatedby reference into the Specification.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to the field of cancer-associatedgenes. Specifically, it relates to novel sequences for use in diagnosisand treatment of cancer and tumors, as well as the use of the novelcompositions in screening methods. The present invention providesmethods of using cancer associated polynucleotides, their correspondinggene products and antibodies specific for the gene products in thedetection, diagnosis, prevention and/or treatment of associated cancers.

BACKGROUND OF THE INVENTION

Oncogenes are genes that can cause cancer. Carcinogenesis can occur by awide variety of mechanisms, including infection of cells by virusescontaining oncogenes, activation of protooncogenes in the host genome,and mutations of protooncogenes and tumor suppressor genes.Carcinogenesis is fundamentally driven by somatic cell evolution (i.e.mutation and natural selection of variants with progressive loss ofgrowth control). The genes that serve as targets for these somaticmutations are classified as either protooncogenes or tumor suppressorgenes, depending on whether their mutant phenotypes are dominant orrecessive, respectively.

There are a number of viruses known to be involved in human cancer aswell as in animal cancer. Of particular interest here are viruses thatdo not contain oncogenes themselves; these are slow-transformingretroviruses. They induce tumors by integrating into the host genome andaffecting neighboring protooncogenes in a variety of ways. Provirusinsertion mutation is a normal consequence of the retroviral life cycle.In infected cells, a DNA copy of the retrovirus genome (called aprovirus) is integrated into the host genome. A newly integratedprovirus can affect gene expression in cis at or near the integrationsite by one of two mechanisms. Type I insertion mutations up-regulatetranscription of proximal genes as a consequence of regulatory sequences(enhancers and/or promoters) within the proviral long terminal repeats(LTRs). Type II insertion mutations cause truncation of coding regionsdue to either integration directly within an open reading frame orintegration within an intron flanked on both sides by coding sequences.The analysis of sequences at or near the insertion sites has led to theidentification of a number of new protooncogenes.

With respect to lymphoma and leukemia, retroviruses such as AKV murineleukemia virus (MLV) or SL3-3 MLV, are potent inducers of tumors wheninoculated into susceptible newborn mice, or when carried in thegermline. A number of sequences have been identified as relevant in theinduction of lymphoma and leukemia by analyzing the insertion sites; seeSorensen et al., J. of Virology 74:2161 (2000); Hansen et al., GenomeRes. 10(2):237-43 (2000); Sorensen et al., J. Virology 70:4063 (1996);Sorensen et al., J. Virology 67:7118 (1993); Joosten et al., Virology268:308 (2000); and Li et al., Nature Genetics 23:348 (1999); all ofwhich are expressly incorporated by reference herein. With respect tocancers, especially breast cancer, prostate cancer and cancers withepithelial origin, the mammalian retrovirus, mouse mammary tumor virus(MMTV) is a potent inducer of tumors when inoculated into susceptiblenewborn mice, or when carried in the germ line. Mammary Tumors in theMouse, edited by J. Hilgers and M. Sluyser; Elsevier/North-HollandBiomedical Press; New York, N.Y.

The pattern of gene expression in a particular living cell ischaracteristic of its current state. Nearly all differences in the stateor type of a cell are reflected in the differences in RNA levels of oneor more genes. Comparing expression patterns of uncharacterized genesmay provide clues to their function. High throughput analysis ofexpression of hundreds or thousands of genes can help in (a)identification of complex genetic diseases, (b) analysis of differentialgene expression over time, between tissues and disease states, and (c)drug discovery and toxicology studies. Increase or decrease in thelevels of expression of certain genes correlate with cancer biology. Forexample, oncogenes are positive regulators of tumorigenesis, while tumorsuppressor genes are negative regulators of tumorigenesis. (Marshall,Cell, 64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991)).

Accordingly, it is an object of the invention to provide polynucleotideand polypeptide sequences involved in cancer and, in particular, inoncogenesis.

Immunotherapy, or the use of antibodies for therapeutic purposes hasbeen used in recent years to treat cancer. Passive immunotherapyinvolves the use of monoclonal antibodies in cancer treatments. See forexample, Cancer: Principles and Practice of Oncology, 6^(th) Edition(2001) Chapt. 20 pp. 495-508. Inherent therapeutic biological activityof these antibodies include direct inhibition of tumor cell growth orsurvival, and the ability to recruit the natural cell killing activityof the body's immune system. These agents are administered alone or inconjunction with radiation or chemotherapeutic agents. Rituxan® andHerceptin®, approved for treatment of lymphoma and breast cancer,respectively, are two examples of such therapeutics. Alternatively,antibodies are used to make antibody conjugates where the antibody islinked to a toxic agent and directs that agent to the tumor byspecifically binding to the tumor. Mylotarg® is an example of anapproved antibody conjugate used for the treatment of leukemia.

Accordingly, it is another object of this invention to provide antigens(cancer-associated polypeptides) associated with a variety of cancers astargets for diagnostic and/or therapeutic antibodies. These antigens arealso useful for drug discovery (e.g., small molecules) and for furthercharacterization of cellular regulation, growth, and differentiation.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods for screening for compositions that modulate cancer,especially lymphoma and leukemia. The present invention also providesmethods for screening for compositions which modulate carcinomas,especially mammary adenocarcinomas. Also provided herein are methods ofinhibiting proliferation of a cell, preferably a lymphoma cell or abreast cancer cell. Methods of treatment of cancer, including diagnosis,are also provided herein.

In one aspect, a method of screening drug candidates comprises providinga cell that expresses a cancer-associated (CA) gene or fragmentsthereof. Preferred embodiments of CA genes are genes that aredifferentially expressed in cancer cells, preferably lymphatic, breast,prostate or epithelial cells, compared to other cells. Preferredembodiments of CA genes used in the methods herein include, but are notlimited to the nucleic acids selected from Tables 1-16 (human genomicsequences of SEQ ID NOS: 4, 10, 18, 26, 34, 44, 50, 60, 82, 88, 112,122, 128, 136, 154, and 170, and sequences of SEQ ID NOS: 5, 11, 13, 19,27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65, 67, 69, 71, 73, 75, 83,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 113, 115, 117, 123, 129,137, 139, 141, 143, 145, 147, 149, 155, 157, 159, 161, 163, 165, 171,and 173 corresponding to the human mRNAs generated therefrom). Themethods further include adding a drug candidate to the cell anddetermining the effect of the drug candidate on the expression of the CAgene.

In one embodiment, the method of screening drug candidates includescomparing the level of expression in the absence of the drug candidateto the level of expression in the presence of the drug candidate.

Also provided herein is a method of screening for a bioactive agentcapable of binding to a CA protein (CAP), the method comprisingcombining the CAP and a candidate bioactive agent, and determining thebinding of the candidate agent to the CAP.

Further provided herein is a method for screening for a bioactive agentcapable of modulating the activity of a CAP. In one embodiment, themethod comprises combining the CAP and a candidate bioactive agent, anddetermining the effect of the candidate agent on the bioactivity of theCAP.

Also provided is a method of evaluating the effect of a candidate cancerdrug comprising administering the drug to a patient and removing a cellsample from the patient. The expression profile of the cell is thendetermined. This method may further comprise comparing the expressionprofile of the patient to an expression profile of a healthy individual.

In a further aspect, a method for inhibiting the activity of a CAprotein is provided. In one embodiment, the method comprisesadministering to a patient an inhibitor of a CA protein preferablyselected from the group consisting of the sequences outlined in Tables1-16 (SEQ ID NOS: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62,64, 66, 68, 70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 114, 116, 118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156,158, 160, 162, 164, 166, 172, and 174).

A method of neutralizing the effect of a CA protein, preferably aprotein encoded by a nucleic acid selected from the group of sequencesoutlined in Tables 1-16 (human genomic sequences of SEQ ID NOS: 4, 10,18, 26, 34, 44, 50, 60, 82, 88, 112, 122, 128, 136, 154, and 170, andsequences of SEQ ID NOS: 5, 11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53,55, 61, 63, 65, 67, 69, 71, 73, 75, 83, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 113, 115, 117, 123, 129, 137, 139, 141, 143, 145, 147,149, 155, 157, 159, 161, 163, 165, 171, and 173 corresponding to thehuman mRNAs generated therefrom), is also provided. Preferably, themethod comprises contacting an agent specific for said protein with saidprotein in an amount sufficient to effect neutralization.

Moreover, provided herein is a biochip comprising a nucleic acid segmentwhich encodes a CA protein, preferably selected from the sequencesoutlined in Tables 1-16 (SEQ ID NOS: 5, 11, 13, 19, 27, 29, 35, 37, 39,45, 51, 53, 55, 61, 63, 65, 67, 69, 71, 73, 75, 83, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 113, 115, 117, 123, 129, 137, 139, 141, 143,145, 147, 149, 155, 157, 159, 161, 163, 165, 171, and 173).

Also provided herein is a method for diagnosing or determining thepropensity to cancers, especially lymphoma or leukemia or carcinoma bysequencing at least one carcinoma or lymphoma gene of an individual. Inyet another aspect of the invention, a method is provided fordetermining cancer including lymphoma and leukemia gene copy numbers inan individual.

The invention provides an isolated nucleic acid comprising at least 10,12, 15, 20 or 30 contiguous nucleotides of a sequence selected from thegroup consisting of the polynucleotide sequences SEQ ID NOS: 5, 11, 13,19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65, 67, 69, 71, 73, 75,83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 113, 115, 117, 123, 129,137, 139, 141, 143, 145, 147, 149, 155, 157, 159, 161, 163, 165, 171,and 173 shown in Tables 1-16, or its complement, or an expression vectorcomprising the isolated nucleic acids and host cells comprising them.

In some embodiments, the polynucleotide, or its complement or a fragmentthereof, further comprises a detectable label, is attached to a solidsupport, is prepared at least in part by chemical synthesis, is anantisense fragment, is single stranded, is double stranded or comprisesa microarray.

The invention provides an isolated polypeptide, encoded within an openreading frame of a CA sequence selected from the group consisting of thepolynucleotide sequences of SEQ ID NOS: 4, 10, 18, 26, 34, 44, 50, 60,82, 88, 112, 122, 128, 136, 154, and 170 shown in Tables 1-16, or itscomplement. The invention provides an isolated polypeptide, wherein saidpolypeptide comprises the amino acid sequence encoded by apolynucleotide selected from the group consisting of SEQ ID NOS: 5, 11,13, 19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65, 67, 69, 71, 73,75, 83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 113, 115, 117, 123,129, 137, 139, 141, 143, 145, 147, 149, 155, 157, 159, 161, 163, 165,171, and 173 shown in Tables 1-16. The invention provides an isolatedpolypeptide, wherein said polypeptide comprises the amino acid sequenceencoded by a polypeptide selected from the group consisting of SEQ IDNOS: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68,70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114,116, 118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160,162, 164, 166, 172, and 174 shown in Tables 1-16.

The invention further provides an isolated polypeptide, comprising theamino acid sequence of an epitope of the amino acid sequence of a CApolypeptide selected from the group consisting of SEQ ID NOS: 6, 12, 14,20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70, 72, 74, 76,84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116, 118, 124,130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162, 164, 166,172, and 174 shown in Tables 1-16, wherein the polypeptide or fragmentthereof may be attached to a solid support. In one embodiment theinvention provides an isolated antibody (monoclonal or polyclonal) orantigen binding fragment thereof, that binds to such a polypeptide. Theisolated antibody or antigen binding fragment thereof may be attached toa solid support, or further comprises a detectable label.

In one embodiment, the invention provides a kit for diagnosing thepresence of cancer in a test sample, said kit comprising at least onepolynucleotide that selectively hybridizes to a CA polynucleotidesequence shown in Tables 1-16, or its complement. In another embodimentthe invention provides an electronic library comprising a CApolynucleotide, a CA polypeptide, or fragment thereof, shown in Tables1-16.

In one embodiment, the invention provides a method of screening foranticancer activity comprising: (a) providing a cell that expresses acancer associated (CA) gene encoded by a nucleic acid sequence selectedfrom the group consisting of the CA sequences shown in Tables 1-16, orfragment thereof; (b) contacting a tissue sample derived from a cancercell with an anticancer drug candidate; (c) monitoring an effect of theanticancer drug candidate on an expression of the CA polynucleotide inthe tissue sample, and optionally (d) comparing the level of expressionin the absence of said drug candidate to the level of expression in thepresence of the drug candidate.

In one embodiment, the invention provides a method for detecting cancerassociated with expression of a polypeptide in a test cell sample,comprising the steps of: (i) detecting a level of expression of at leastone polypeptide selected from the group consisting of SEQ ID NOS: 6, 12,14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70, 72, 74,76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116, 118, 124,130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162, 164, 166,172, and 174 shown in Tables 1-16, or a fragment thereof; and (ii)comparing the level of expression of the polypeptide in the test samplewith a level of expression of polypeptide in a normal cell sample,wherein an altered level of expression of the polypeptide in the testcell sample relative to the level of polypeptide expression in thenormal cell sample is indicative of the presence of cancer in the testcell sample.

In another embodiment, the invention provides a method for detectingcancer associated with expression of a polypeptide in a test cellsample, comprising the steps of: (i) detecting a level of activity of atleast one polypeptide selected from the group consisting of SEQ ID NOS:6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70,72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116,118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162,164, 166, 172, and 174 shown in Tables 1-16, or a fragment thereof,wherein said activity corresponds to at least one activity for thepolypeptide listed in Table 18; and (ii) comparing the level of activityof the polypeptide in the test sample with a level of activity ofpolypeptide in a normal cell sample, wherein an altered level ofactivity of the polypeptide in the test cell sample relative to thelevel of polypeptide activity in the normal cell sample is indicative ofthe presence of cancer in the test cell sample.

In another embodiment, the invention provides a method for detectingcancer associated with the presence of an antibody in a test serumsample, comprising the steps of: (i) detecting a level of an antibodyagainst an antigenic polypeptide selected from the group consisting ofSEQ ID NOS: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64,66, 68, 70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,114, 116, 118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158,160, 162, 164, 166, 172, and 174 shown in Tables 1-16, or antigenicfragment thereof; and (ii) comparing said level of said antibody in thetest sample with a level of said antibody in the control sample, whereinan altered level of antibody in said test sample relative to the levelof antibody in the control sample is indicative of the presence ofcancer in the test serum sample.

The invention provides a method for screening for a bioactive agentcapable of modulating the activity of a CA protein (CAP), wherein saidCAP is encoded by a nucleic acid comprising a nucleic acid sequenceselected from the group consisting of the polynucleotide sequences SEQID NOS: 5, 11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65,67, 69, 71, 73, 75, 83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 113,115, 117, 123, 129, 137, 139, 141, 143, 145, 147, 149, 155, 157, 159,161, 163, 165, 171, and 173 shown in Tables 1-16, said methodcomprising: a) combining said CAP and a candidate bioactive agent; andb) determining the effect of the candidate agent on the bioactivity ofsaid CAP. According to the method the bioactive agent: affects theexpression of the CA protein (CAP); affects the activity of the CAprotein (CAP), wherein such activity is selected from the activitieslisted in Table 18.

In one embodiment, the invention provides a method for diagnosing cancercomprising: a) determining the expression of one or more genescomprising a nucleic acid sequence selected from the group consisting ofthe human genomic and mRNA sequences outlined in Tables 1-16, in a firsttissue type of a first individual; and b) comparing said expression ofsaid gene(s) from a second normal tissue type from said first individualor a second unaffected individual; wherein a difference in saidexpression indicates that the first individual has cancer.

In another embodiment the invention provides a method for treatingcancers comprising administering to a patient a bioactive agentmodulating the activity of a CA protein (CAP), wherein said CAP isencoded by a nucleic acid comprising a nucleic acid sequence selectedfrom the group consisting of the human nucleic acid sequences in Tables1-16 and further wherein the bioactive agent binds to the CA protein,wherein the CA protein: is a signalling protein wherein the CAP sequenceis selected from the group consisting of SEQ ID NOS: 6, 12, 14, 96, 130,172 and 174 shown in Tables 1-16; is involved in signal transductionwherein the CAP sequence is selected from the group consisting of SEQ IDNOS: 20, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, and 108; is a celladhesion protein wherein the CAP sequence is selected from the groupconsisting of SEQ ID NOS: 36, 38, 40, 84, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 124, 172, and 174; is involved in inflammatory responsewherein the CAP sequence is selected from the group consisting of SEQ IDNOS: 62, 64, 66, 68, 72, 74, and 76; is involved in pheromone responsewherein the CAP sequence is selected from the group consisting of SEQ IDNOS: 138, 140, 142, 144, 146, 148, and 150; is an ion transport proteinwherein the CAP sequence is selected from the group consisting of SEQ IDNOS: 28, 30, 114, and 118 as shown in Tables 1-16.

The invention provides monoclonal antibodies that preferentially bindsto a CA protein (CAP) that is expressed on a cell surface, wherein theCA protein selected from the group consisting of SEQ ID NOS: 6, 12, 14,20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70, 72, 74, 76,84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116, 118, 124,130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162, 164, 166,172, and 174; preferably to the extracellular domain of the CA protein;preferably to a CA protein differentially expressed on a cancer cellsurface relative to a normal cell surface or preferably to at least onehuman cancer cell line; preferably linked to a therapeutic agent; orpreferably humanized. Kits and pharmaceutical compositions for detectinga presence or an absence of cancer cells in an individual, andcomprising such antibodies are also provided.

The invention also provides a method for detecting a presence or anabsence of cancer cells in an individual, the method comprising:contacting cells from the individual with the antibody according to theinvention; and detecting a complex of a CAP from the cancer cells andthe antibody, wherein detection of the complex correlates with thepresence of cancer cells in the individual. In one embodiment theinvention provides a method for inhibiting growth of cancer cells in anindividual, the method comprising: administering to the individual aneffective amount of a pharmaceutical composition according to theinvention. In another embodiment the invention provides a method fordelivering a therapeutic agent to cancer cells in an individual, themethod comprising: administering to the individual an effective amountof a pharmaceutical composition according to according to the invention.

Novel sequences associated with cancer are also provided herein. Otheraspects of the invention will become apparent to the skilled artisan bythe following description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts PCR amplification of host-provirus junction fragments.

FIG. 2 shows an example of average threshold cycle (C_(T)) values for ahousekeeper gene and target gene.

FIG. 3 shows an example of the calculated difference (ΔΔC_(T)) betweenthe C_(T) values of target and housekeeper genes (ΔC_(T)) for varioussamples.

FIG. 4 shows the ΔΔC_(T) and comparative expression level for eachsample from FIG. 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to a number of sequences associatedwith cancers, especially lymphoma, breast cancer or prostate cancer. Therelatively tight linkage between clonally-integrated proviruses andprotooncogenes forms “provirus tagging”, in which slow-transformingretroviruses that act by an insertion mutation mechanism are used toisolate protooncogenes. In some models, uninfected animals have lowcancer rates, and infected animals have high cancer rates. It is knownthat many of the retroviruses involved do not carry transduced hostprotooncogenes or pathogenic trans-acting viral genes, and thus thecancer incidence must therefore be a direct consequence of proviralintegration effects into host protooncogenes. Since proviral integrationis random, rare integrants will “activate” host protooncogenes thatprovide a selective growth advantage, and these rare events result innew proviruses at clonal stoichiometries in tumors. In contrast tomutations caused by chemicals, radiation, or spontaneous errors,protooncogene insertion mutations can be easily located by virtue of thefact that a convenient-sized genetic marker of known sequence (theprovirus) is present at the site of mutation. Host sequences that flankclonally integrated proviruses can be cloned using a variety ofstrategies. Once these sequences are in hand, the tagged protooncogenescan be subsequently identified. The presence of provirus at the samelocus in two or more independent tumors is prima facie evidence that aprotooncogene is present at or very near the provirus integration sites.This is because the genome is too large for random integrations toresult in observable clustering. Any clustering that is detected isunequivocal evidence for biological selection (i.e. the tumorphenotype). Moreover, the pattern of proviral integrants (includingorientations) provides compelling positional information that makeslocalization of the target gene at each cluster relatively simple. Thethree mammalian retroviruses that are known to cause cancer by aninsertion mutation mechanism are FeLV (leukemia/lymphoma in cats), MLV(leukemia/lymphoma in mice and rats), and MMTV (mammary cancer in mice).

Thus, the use of oncogenic retroviruses, whose sequences insert into thegenome of the host organism resulting in cancer, allows theidentification of host sequences involved in cancer. These sequences maythen be used in a number of different ways, including diagnosis,prognosis, screening for modulators (including both agonists andantagonists), antibody generation (for immunotherapy and imaging), etc.However, as will be appreciated by those in the art, oncogenes that areidentified in one type of cancer such as lymphoma or leukemia have astrong likelihood of being involved in other types of cancers as well.Thus, while the sequences outlined herein are initially identified ascorrelated with lymphoma, they can also be found in other types ofcancers as well, outlined below.

DEFINITIONS

Accordingly, the present invention provides nucleic acid and proteinsequences that are associated with cancer, herein termed “cancerassociated” or “CA” sequences. In one embodiment, the present inventionprovides nucleic acid and protein sequences that are associated withcancers that originate in lymphatic tissue, herein termed “lymphomaassociated,” “leukemia associated” or “LA” sequences. In anotherembodiment, the present invention provides nucleic acid and proteinsequences that are associated with carcinomas which originate in breasttissue, herein termed “breast cancer associated” or “BC” sequences.

Suitable cancers that can be diagnosed or screened for using the methodsof the present invention include cancers classified by site or byhistological type. Cancers classified by site include cancer of the oralcavity and pharynx (lip, tongue, salivary gland, floor of mouth, gum andother mouth, nasopharynx, tonsil, oropharynx, hypopharynx, otheroral/pharynx); cancers of the digestive system (esophagus; stomach;small intestine; colon and rectum; anus, anal canal, and anorectum;liver; intrahepatic bile duct; gallbladder; other biliary; pancreas;retroperitoneum; peritoneum, omentum, and mesentery; other digestive);cancers of the respiratory system (nasal cavity, middle ear, andsinuses; larynx; lung and bronchus; pleura; trachea, mediastinum, andother respiratory); cancers of the mesothelioma; bones and joints; andsoft tissue, including heart; skin cancers, including melanomas andother non-epithelial skin cancers; Kaposi's sarcoma and breast cancer;cancer of the female genital system (cervix uteri; corpus uteri; uterus,nos; ovary; vagina; vulva; and other female genital); cancers of themale genital system (prostate gland; testis; penis; and other malegenital); cancers of the urinary system (urinary bladder; kidney andrenal pelvis; ureter; and other urinary); cancers of the eye and orbit;cancers of the brain and nervous system (brain; and other nervoussystem); cancers of the endocrine system (thyroid gland and otherendocrine, including thymus); lymphomas (Hodgkin's disease andnon-Hodgkin's lymphoma), multiple myelonia, and leukemias (lymphocyticleukemia; myeloid leukemia; monocytic leukemia; and other leukemias).

Other cancers, classified by histological type, that may be associatedwith the sequences of the invention include, but are not limited to,Neoplasm, malignant; Carcinoma, NOS; Carcinoma, undifferentiated, NOS;Giant and spindle cell carcinoma; Small cell carcinoma, NOS; Papillarycarcinoma, NOS; Squamous cell carcinoma, NOS; Lymphoepithelialcarcinoma; Basal cell carcinoma, NOS; Pilomatrix carcinoma; Transitionalcell carcinoma, NOS; Papillary transitional cell carcinoma;Adenocarcinoma, NOS; Gastrinoma, malignant; Cholangiocarcinoma;Hepatocellular carcinoma, NOS; Combined hepatocellular carcinoma andcholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cystic carcinoma;Adenocarcinoma in adenomatous polyp; Adenocarcinoma, familial polyposiscoli; Solid carcinoma, NOS; Carcinoid tumor, malignant;Bronchiolo-alveolar adenocarcinoma; Papillary adenocarcinoma, NOS;Chromophobe carcinoma; Acidophil carcinoma; Oxyphilic adenocarcinoma;Basophil carcinoma; Clear cell adenocarcinoma, NOS; Granular cellcarcinoma; Follicular adenocarcinoma, NOS; Papillary and follicularadenocarcinoma; Nonencapsulating sclerosing carcinoma; Adrenal corticalcarcinoma; Endometroid carcinoma; Skin appendage carcinoma; Apocrineadenocarcinoma; Sebaceous adenocarcinoma; Ceruminous adenocarcinoma;Mucoepidermoid carcinoma; Cystadenocarcinoma, NOS; Papillarycystadenocarcinoma, NOS; Papillary serous cystadenocarcinoma; Mucinouscystadenocarcinoma, NOS; Mucinous adenocarcinoma; Signet ring cellcarcinoma; Infiltrating duct carcinoma; Medullary carcinoma, NOS;Lobular carcinoma; Inflammatory carcinoma; Paget's disease, mammary;Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma w/squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor,malignant; Thecoma, malignant; Granulosa cell tumor, malignant;Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor,malignant; Lipid cell tumor, malignant; Paraganglioma, malignant;Extra-mammary paraganglioma, malignant; Pheochromocytoma;Glomangiosarcoma; Malignant melanoma, NOS; Amelanotic melanoma;Superficial spreading melanoma; Malig melanoma in giant pigmented nevus;Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma, NOS;Fibrosarcoma, NOS; Fibrous histiocytoma, malignant; Myxosarcoma;Liposarcoma, NOS; Leiomyosarcoma, NOS; Rhabdomyosarcoma, NOS; Embryonalrhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma, NOS; Mixedtumor, malignant, NOS; Mullerian mixed tumor; Nephroblastoma;Hepatoblastoma; Carcinosarcoma, NOS; Mesenchymoma, malignant; Brennertumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma, NOS;Mesothelioma, malignant; Dysgerminoma; Embryonal carcinoma, NOS;Teratoma, malignant, NOS; Struma ovarii, malignant; Choriocarcinoma;Mesonephroma, malignant; Hemangiosarcoma; Hemangioendothelioma,malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant;Lymphangiosarcoma; Osteosarcoma, NOS; Juxtacortical osteosarcoma;Chondrosarcoma, NOS; Chondroblastoma, malignant; Mesenchymalchondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenictumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant;Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma,malignant; Ependymoma, NOS; Astrocytoma, NOS; Protoplasmic astrocytoma;Fibrillary astrocytoma; Astroblastoma; Glioblastoma, NOS;Oligodendroglioma, NOS; Oligodendroblastoma; Primitive neuroectodermal;Cerebellar sarcoma, NOS; Ganglioneuroblastoma; Neuroblastoma, NOS;Retinoblastoma, NOS; Olfactory neurogenic tumor; Meningioma, malignant;Neurofibrosarcoma; Neurilemmoma, malignant; Granular cell tumor,malignant; Malignant lymphoma, NOS; Hodgkin's disease, NOS; Hodgkin's;paragranuloma, NOS; Malignant lymphoma, small lymphocytic; Malignantlymphoma, large cell, diffuse; Malignant lymphoma, follicular, NOS;Mycosis fungoides; Other specified non-Hodgkin's lymphomas; Malignanthistiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferativesmall intestinal disease; Leukemia, NOS; Lymphoid leukemia, NOS; Plasmacell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloidleukemia, NOS; Basophilic leukemia; Eosinophilic leukemia; Monocyticleukemia, NOS; Mast cell leukemia; Megakaryoblastic leukemia; Myeloidsarcoma; and Hairy cell leukemia.

In addition, the CA genes may be involved in other diseases such as, butnot limited to, diseases associated with aging or neurodegeneration.

“Association” in this context means that the nucleotide or proteinsequences are either differentially expressed, activated, inactivated oraltered in cancers as compared to normal tissue. As outlined below, CAsequences include those that are up-regulated (i.e. expressed at ahigher level), as well as those that are down-regulated (i.e. expressedat a lower level), in cancers. CA sequences also include sequences thathave been altered (i.e., truncated sequences or sequences withsubstitutions, deletions or insertions, including point mutations) andshow either the same expression profile or an altered profile. In apreferred embodiment, the CA sequences are from humans; however, as willbe appreciated by those in the art, CA sequences from other organismsmay be useful in animal models of disease and drug evaluation; thus,other CA sequences are provided, from vertebrates, including mammals,including rodents (rats, mice, hamsters, guinea pigs, etc.), primates,and farm animals (including sheep, goats, pigs, cows, horses, etc). Insome cases, prokaryotic CA sequences may be useful. CA sequences fromother organisms may be obtained using the techniques outlined below.

CA sequences include both nucleic acid and amino acid sequences. In apreferred embodiment, the CA sequences are recombinant nucleic acids. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid by polymerases and endonucleases, in a form not normally found innature. Thus a recombinant nucleic acid is also an isolated nucleicacid, in a linear form, or cloned in a vector formed in vitro byligating DNA molecules that are not normally joined, are both consideredrecombinant for the purposes of this invention. It is understood thatonce a recombinant nucleic acid is made and reintroduced into a hostcell or organism, it will replicate using the in vivo cellular machineryof the host cell rather than in vitro manipulations; however, suchnucleic acids, once produced recombinantly, although subsequentlyreplicated in vivo, are still considered recombinant or isolated for thepurposes of the invention. As used herein a “polynucleotide” or “nucleicacid” is a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. This term refers only to theprimary structure of the molecule. Thus, this term includes double- andsingle-stranded DNA and RNA. It also includes known types ofmodifications, for example, labels which are known in the art,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, etc.), those containing alkylators, those withmodified linkages (e.g., alpha anomeric nucleic acids, etc.), as well asunmodified forms of the polynucleotide.

As used herein, a polynucleotide “derived from” a designated sequencerefers to a polynucleotide sequence which is comprised of a sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding to aregion of the designated nucleotide sequence. “Corresponding” meanshomologous to or complementary to the designated sequence. Preferably,the sequence of the region from which the polynucleotide is derived ishomologous to or complementary to a sequence that is unique to a CAgene.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesabout 50-75% by weight of the total protein, with about 80% beingpreferred, and about 90% being particularly preferred. The definitionincludes the production of a CA protein from one organism in a differentorganism or host cell. Alternatively, the protein may be made at asignificantly higher concentration than is normally seen, through theuse of an inducible promoter or high expression promoter, such that theprotein is made at increased concentration levels. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions, as discussed below.

In a preferred embodiment, the CA sequences are nucleic acids. As willbe appreciated by those in the art and is more fully outlined below, CAsequences are useful in a variety of applications, including diagnosticapplications, which will detect naturally occurring nucleic acids, aswell as screening applications; for example, biochips comprising nucleicacid probes to the CA sequences can be generated. In the broadest sense,use of “nucleic acid,” “polynucleotide” or “oligonucleotide” orequivalents herein means at least two nucleotides covalently linkedtogether. In some embodiments, an oligonucleotide is an oligomer of 6,8, 10, 12, 20, 30 or up to 100 nucleotides. A “polynucleotide” or“oligonucleotide” may comprise DNA, RNA, PNA or a polymer of nucleotideslinked by phosphodiester and/or any alternate bonds.

A nucleic acid of the present invention generally containsphosphodiester bonds, although in some cases, as outlined below (forexample, in antisense applications or when a nucleic acid is a candidatedrug agent), nucleic acid analogs may have alternate backbones,comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem.Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988);and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321(1989), O-methylphosphoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Otheranalog nucleic acids include those with positive backbones (Denpcy etal., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within one definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done for a varietyof reasons, for example to increase the stability and half-life of suchmolecules in physiological environments for use in anti-senseapplications or as probes on a biochip.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. As will be appreciated by those in the art, thedepiction of a single strand “Watson” also defines the sequence of theother strand “Crick”; thus the sequences described herein also includesthe complement of the sequence. The nucleic acid may be DNA, bothgenomic and cDNA, RNA, or a hybrid, where the nucleic acid contains anycombination of deoxyribo- and ribo-nucleotides, and any combination ofbases, including uracil, adenine, thymine, cytosine, guanine, inosine,xanthine, hypoxanthine, isocytosine, isoguanine, etc. As used herein,the term “nucleoside” includes nucleotides and nucleoside and nucleotideanalogs, and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

As used herein, the term “tag,” “sequence tag” or “primer tag sequence”refers to an oligonucleotide with specific nucleic acid sequence thatserves to identify a batch of polynucleotides bearing such tags therein.Polynucleotides from the same biological source are covalently taggedwith a specific sequence tag so that in subsequent analysis thepolynucleotide can be identified according to its source of origin. Thesequence tags also serve as primers for nucleic acid amplificationreactions.

A “microarray” is a linear or two-dimensional array of preferablydiscrete regions, each having a defined area, formed on the surface of asolid support. The density of the discrete regions on a microarray isdetermined by the total numbers of target polynucleotides to be detectedon the surface of a single solid phase support, preferably at leastabout 50/cm², more preferably at least about 100/cm², even morepreferably at least about 500/cm², and still more preferably at leastabout 1,000/cm². As used herein, a DNA microarray is an array ofoligonucleotide primers placed on a chip or other surfaces used toamplify or clone target polynucleotides. Since the position of eachparticular group of primers in the array is known, the identities of thetarget polynucleotides can be determined based on their binding to aparticular position in the microarray.

A “linker” is a synthetic oligodeoxyribonucleotide that contains arestriction site. A linker may be blunt end-ligated onto the ends of DNAfragments to create restriction sites that can be used in the subsequentcloning of the fragment into a vector molecule.

The term “label” refers to a composition capable of producing adetectable signal indicative of the presence of the targetpolynucleotide in an assay sample. Suitable labels includeradioisotopes, nucleotide chromophores, enzymes, substrates, fluorescentmolecules, chemiluminescent moieties, magnetic particles, bioluminescentmoieties, and the like. As such, a label is any composition detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical, or any other appropriate means. The term“label” is used to refer to any chemical group or moiety having adetectable physical property or any compound capable of causing achemical group or moiety to exhibit a detectable physical property, suchas an enzyme that catalyzes conversion of a substrate into a detectableproduct. The term “label” also encompasses compounds that inhibit theexpression of a particular physical property. The label may also be acompound that is a member of a binding pair, the other member of whichbears a detectable physical property.

The term “support” refers to conventional supports such as beads,particles, dipsticks, fibers, filters, membranes, and silane or silicatesupports such as glass slides.

The term “amplify” is used in the broad sense to mean creating anamplification product which may include, for example, additional targetmolecules, or target-like molecules or molecules complementary to thetarget molecule, which molecules are created by virtue of the presenceof the target molecule in the sample. In the situation where the targetis a nucleic acid, an amplification product can be made enzymaticallywith DNA or RNA polymerases or reverse transcriptases.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from an individual, including but not limited to, forexample, blood, plasma, serum, spinal fluid, lymph fluid, skin,respiratory, intestinal and genitourinary tracts, tears, saliva, milk,cells (including but not limited to blood cells), tumors, organs, andalso samples of in vitro cell culture constituents.

The term “biological sources” as used herein refers to the sources fromwhich the target polynucleotides are derived. The source can be of anyform of “sample” as described above, including but not limited to, cell,tissue or fluid. “Different biological sources” can refer to differentcells/tissues/organs of the same individual, or cells/tissues/organsfrom different individuals of the same species, or cells/tissues/organsfrom different species.

Cancer-Associated Sequences

The CA sequences of the invention were initially identified by infectionof mice with a retrovirus such as murine leukemia virus (MLV) resultingin lymphoma. Retroviruses have a genome that is made out of RNA. After aretrovirus infects a host cell, a double stranded DNA copy of theretrovirus genome (a “provirus”) is inserted into the genomic DNA of thehost cell. The integrated provirus may affect the expression of hostgenes at or near the site of integration—a phenomenon known asretroviral insertional mutagenesis. Possible changes in the expressionof host cell genes include: (i) increased expression of genes near thesite of integration resulting from the proximity of elements in theprovirus that act as transcriptional promoters and enhancers, (ii)functional inactivation of a gene caused by the integration of aprovirus into the gene itself thus preventing the synthesis of afunctional gene product, or (iii) expression of a mutated protein thathas a different activity to the normal protein. Typically such a proteinwould be prematurely truncated and lack a regulatory domain near the Cterminus. Such a protein might be constitutively active, or act as adominant negative inhibitor of the normal protein. For example,retrovirus enhancers, including that of SL3-3, are known to act on genesup to approximately 200 kilobases from the insertion site. Moreover,many of these sequences are also involved in other cancers and diseasestates. Sequences of mouse genes according to this invention, that areidentified in this manner are shown as mDxx-yyy in Tables 1-16.

A CA sequence can be initially identified by substantial nucleic acidand/or amino acid sequence homology to the CA sequences outlined herein.Such homology can be based upon the overall nucleic acid or amino acidsequence, and is generally determined as outlined below, using eitherhomology programs or hybridization conditions.

In one embodiment, CA sequences are those that are up-regulated incancers; that is, the expression of these genes is higher in cancertissue as compared to normal tissue of the same differentiation stage.“Up-regulation” as used herein means increased expression by about 50%,preferably about 100%, more preferably about 150% to about 200%, withup-regulation from 300% to 1000% being preferred.

In another embodiment, CA sequences are those that are down-regulated incancers; that is, the expression of these genes is lower in cancertissue as compared to normal tissue of the same differentiation stage.“Down-regulation” as used herein means decreased expression by about50%, preferably about 100%, more preferably about 150% to about 200%,with down-regulation from 300% to 1000% to no expression beingpreferred.

In yet another embodiment, CA sequences are those that have alteredsequences but show either the same or an altered expression profile ascompared to normal lymphoid tissue of the same differentiation stage.“Altered CA sequences” as used herein also refers to sequences that aretruncated, contain insertions or contain point mutations.

CA proteins of the present invention may be classified as secretedproteins, transmembrane proteins or intracellular proteins. In apreferred embodiment the CA protein is an intracellular protein.Intracellular proteins may be found in the cytoplasm and/or in thenucleus. Intracellular proteins are involved in all aspects of cellularfunction and replication (including, for example, signaling pathways);aberrant expression of such proteins results in unregulated ordisregulated cellular processes. For example, many intracellularproteins have enzymatic activity such as protein kinase activity,protein phosphatase activity, protease activity, nucleotide cyclaseactivity, polymerase activity and the like. Intracellular proteins alsoserve as docking proteins that are involved in organizing complexes ofproteins, or targeting proteins to various subcellular localizations,and are involved in maintaining the structural integrity of organelles.

An increasingly appreciated concept in characterizing intracellularproteins is the presence in the proteins of one or more motifs for whichdefined functions have been attributed. In addition to the highlyconserved sequences found in the enzymatic domain of proteins, highlyconserved sequences have been identified in proteins that are involvedin protein-protein interaction. For example, Src-homology-2 (SH2)domains bind tyrosine-phosphorylated targets in a sequence dependentmanner. PTB domains, which are distinct from SH2 domains, also bindtyrosine phosphorylated targets. SH3 domains bind to proline-richtargets. In addition, PH domains, tetratricopeptide repeats and WDdomains to name only a few, have been shown to mediate protein-proteininteractions. Some of these may also be involved in binding tophospholipids or other second messengers. As will be appreciated by oneof ordinary skill in the art, these motifs can be identified on thebasis of primary sequence; thus, an analysis of the sequence of proteinsmay provide insight into both the enzymatic potential of the moleculeand/or molecules with which the protein may associate.

In a preferred embodiment, the CA sequences are transmembrane proteins.Transmembrane proteins are molecules that span the phospholipid bilayerof a cell. They may have an intracellular domain, an extracellulardomain, or both. The intracellular domains of such proteins may have anumber of functions including those already described for intracellularproteins. For example, the intracellular domain may have enzymaticactivity and/or may serve as a binding site for additional proteins.Frequently the intracellular domain of transmembrane proteins servesboth roles. For example certain receptor tyrosine kinases have bothprotein kinase activity and SH2 domains. In addition,autophosphorylation of tyrosines on the receptor molecule itself createsbinding sites for additional SH2 domain containing proteins.

Transmembrane proteins may contain from one to many transmembranedomains. For example, receptor tyrosine kinases, certain cytokinereceptors, receptor guanylyl cyclases and receptor serine/threonineprotein kinases contain a single transmembrane domain. However, variousother proteins including channels and adenylyl cyclases contain numeroustransmembrane domains. Many important cell surface receptors areclassified as “seven transmembrane domain” proteins, as they contain 7membrane spanning regions. Important transmembrane protein receptorsinclude, but are not limited to insulin receptor, insulin-like growthfactor receptor, human growth hormone receptor, glucose transporters,transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, leptin receptor, interleukin receptors, e.g. IL-1receptor, IL-2 receptor, etc. CA proteins may be derived from genes thatregulate apoptosis (IL-3, GM-CSF and Bcl-x) or are shown to have a rolein the regulation of apoptosis.

Characteristics of transmembrane domains include approximately 20consecutive hydrophobic amino acids that may be followed by chargedamino acids. Therefore, upon analysis of the amino acid sequence of aparticular protein, the localization and number of transmembrane domainswithin the protein may be predicted.

The extracellular domains of transmembrane proteins are diverse;however, conserved motifs are found repeatedly among variousextracellular domains. Conserved structure and/or functions have beenascribed to different extracellular motifs. For example, cytokinereceptors are characterized by a cluster of cysteines and a WSXWS(W=tryptophan, S=serine, X=any amino acid) motif. Immunoglobulin-likedomains are highly conserved. Mucin-like domains may be involved in celladhesion and leucine-rich repeats participate in protein-proteininteractions.

Many extracellular domains are involved in binding to other molecules.In one aspect, extracellular domains are receptors. Factors that bindthe receptor domain include circulating ligands, which may be peptides,proteins, or small molecules such as adenosine and the like. Forexample, growth factors such as EGF, FGF and PDGF are circulating growthfactors that bind to their cognate receptors to initiate a variety ofcellular responses. Other factors include cytokines, mitogenic factors,neurotrophic factors and the like. Extracellular domains also bind tocell-associated molecules. In this respect, they mediate cell-cellinteractions. Cell-associated ligands can be tethered to the cell forexample via a glycosylphosphatidylinositol (GPI) anchor, or maythemselves be transmembrane proteins. Extracellular domains alsoassociate with the extracellular matrix and contribute to themaintenance of the cell structure.

CA proteins that are transmembrane are particularly preferred in thepresent invention as they are good targets for immunotherapeutics, asare described herein. In addition, as outlined below, transmembraneproteins can be also useful in imaging modalities.

It will also be appreciated by those in the art that a transmembraneprotein can be made soluble by removing transmembrane sequences, forexample through recombinant methods. Furthermore, transmembrane proteinsthat have been made soluble can be made to be secreted throughrecombinant means by adding an appropriate signal sequence.

In a preferred embodiment, the CA proteins are secreted proteins; thesecretion of which can be either constitutive or regulated. Theseproteins have a signal peptide or signal sequence that targets themolecule to the secretory pathway. Secreted proteins are involved innumerous physiological events; by virtue of their circulating nature,they serve to transmit signals to various other cell types. The secretedprotein may function in an autocrine manner (acting on the cell thatsecreted the factor), a paracrine manner (acting on cells in closeproximity to the cell that secreted the factor) or an endocrine manner(acting on cells at a distance). Thus secreted molecules find use inmodulating or altering numerous aspects of physiology. CA proteins thatare secreted proteins are particularly preferred in the presentinvention as they serve as good targets for diagnostic markers, forexample for blood tests.

CA Sequences and Homologs

A CA sequence is initially identified by substantial nucleic acid and/oramino acid sequence homology to the CA sequences outlined herein. Suchhomology can be based upon the overall nucleic acid or amino acidsequence, and is generally determined as outlined below, using eitherhomology programs or hybridization conditions.

As used herein, a nucleic acid is a “CA nucleic acid” if the overallhomology of the nucleic acid sequence to one of the nucleic acids ofTables 1-16 is preferably greater than about 75%, more preferablygreater than about 80%, even more preferably greater than about 85% andmost preferably greater than 90%. In some embodiments the homology willbe as high as about 93 to 95 or 98%. In a preferred embodiment, thesequences that are used to determine sequence identity or similarity areselected from those of the nucleic acids of Tables 1-16. In anotherembodiment, the sequences are naturally occurring allelic variants ofthe sequences of the nucleic acids of Tables 1-16. In anotherembodiment, the sequences are sequence variants as further describedherein.

Homology in this context means sequence similarity or identity, withidentity being preferred. A preferred comparison for homology purposesis to compare the sequence containing sequencing errors to the correctsequence. This homology will be determined using standard techniquesknown in the art, including, but not limited to, the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, PNASUSA 85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.), theBest Fit sequence program described by Devereux et al., Nucl. Acid Res.12:387-395 (1984), preferably using the default settings, or byinspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters include adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST (Basic LocalAlignment Search Tool) algorithm, described in Altschul et al., J. Mol.Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787(1993). A particularly useful BLAST program is the WU-BLAST-2 programwhich was obtained from Altschul et al., Methods in Enzymology, 266:460-480 (1996); http://blast.wustl.edu/]. WU-BLAST-2 uses several searchparameters, most of which are set to the default values. The adjustableparameters are set with the following values: overlap span=1, overlapfraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parametersare dynamic values and are established by the program itself dependingupon the composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values may be adjusted to increase sensitivity. Apercent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

Thus, “percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues of the nucleic acids of Tables1-16. A preferred method utilizes the BLASTN module of WU-BLAST-2 set tothe default parameters, with overlap span and overlap fraction set to 1and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleotides than those of the nucleic acids of Tables 1-16, it isunderstood that the percentage of homology will be determined based onthe number of homologous nucleosides in relation to the total number ofnucleosides. Thus homology of sequences shorter than those of thesequences identified herein will be determined using the number ofnucleosides in the shorter sequence.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC (“salinesodium citrate”; 9 mM NaCl, 0.9 mM sodium citrate), 0.5% SDS, 1.0 mMEDTA (pH 8.0); hybridizing at 50-60° C., 5×SSC, overnight; followed bywashing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. One skilled in the art will understand that thestringency of hybridization can be readily manipulated, such as byaltering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C., or 65-70° C.Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide.

Thus nucleic acids that hybridize under high stringency to the nucleicacids identified in the figures, or their complements, are considered CAsequences. High stringency conditions are known in the art; see forexample Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal., both of which are hereby incorporated by reference. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for longer probes (e.g. greater than 50 nucleotides). In anotherembodiment, less stringent hybridization conditions are used; forexample, moderate or low stringency conditions may be used, as are knownin the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

In addition, the CA nucleic acid sequences of the invention arefragments of larger genes, i.e. they are nucleic acid segments.Alternatively, the CA nucleic acid sequences can serve as indicators ofoncogene position, for example, the CA sequence may be an enhancer thatactivates a protooncogene. “Genes” in this context includes codingregions, non-coding regions, and mixtures of coding and non-codingregions. Accordingly, as will be appreciated by those in the art, usingthe sequences provided herein, additional sequences of the CA genes canbe obtained, using techniques well known in the art for cloning eitherlonger sequences or the full-length sequences; see Maniatis et al., andAusubel, et al., supra, hereby expressly incorporated by reference. Ingeneral, this is done using PCR, for example, kinetic PCR.

Detection of CA Expression

Once the CA nucleic acid is identified, it can be cloned and, ifnecessary, its constituent parts recombined to form the entire CAnucleic acid. Once isolated from its natural source, e.g., containedwithin a plasmid or other vector or excised therefrom as a linearnucleic acid segment, the recombinant CA nucleic acid can be furtherused as a probe to identify and isolate other CA nucleic acids, forexample additional coding regions. It can also be used as a “precursor”nucleic acid to make modified or variant CA nucleic acids and proteins.In a preferred embodiment, once a CA gene is identified its nucleotidesequence is used to design probes specific for the CA gene.

The CA nucleic acids of the present invention are used in several ways.In a first embodiment, nucleic acid probes hybridizable to CA nucleicacids are made and attached to biochips to be used in screening anddiagnostic methods, or for gene therapy and/or antisense applications.Alternatively, the CA nucleic acids that include coding regions of CAproteins can be put into expression vectors for the expression of CAproteins, again either for screening purposes or for administration to apatient.

Recent developments in DNA microarray technology make it possible toconduct a large scale assay of a plurality of target CA nucleic acidmolecules on a single solid phase support. U.S. Pat. No. 5,837,832 (Cheeet al.) and related patent applications describe immobilizing an arrayof oligonucleotide probes for hybridization and detection of specificnucleic acid sequences in a sample. Target polynucleotides of interestisolated from a tissue of interest are hybridized to the DNA chip andthe specific sequences detected based on the target polynucleotides'preference and degree of hybridization at discrete probe locations. Oneimportant use of arrays is in the analysis of differential geneexpression, where the profile of expression of genes in different cells,often a cell of interest and a control cell, is compared and anydifferences in gene expression among the respective cells areidentified. Such information is useful for the identification of thetypes of genes expressed in a particular cell or tissue type anddiagnosis of cancer conditions based on the expression profile.

Typically, RNA from the sample of interest is subjected to reversetranscription to obtain labeled cDNA. See U.S. Pat. No. 6,410,229(Lockhart et al.) The cDNA is then hybridized to oligonucleotides orcDNAs of known sequence arrayed on a chip or other surface in a knownorder. The location of the oligonucleotide to which the labeled cDNAhybridizes provides sequence information on the cDNA, while the amountof labeled hybridized RNA or cDNA provides an estimate of the relativerepresentation of the RNA or cDNA of interest. See Schena, et al.Science 270:467-470 (1995). For example, use of a cDNA microarray toanalyze gene expression patterns in human cancer is described by DeRisi,et al. (Nature Genetics 14:457-460 (1996)).

In a preferred embodiment, nucleic acid probes corresponding to CAnucleic acids (both the nucleic acid sequences outlined in the figuresand/or the complements thereof) are made. Typically, these probes aresynthesized based on the disclosed sequences of this invention. Thenucleic acid probes attached to the biochip are designed to besubstantially complementary to the CA nucleic acids, i.e. the targetsequence (either the target sequence of the sample or to other probesequences, for example in sandwich assays), such that specifichybridization of the target sequence and the probes of the presentinvention occurs. As outlined below, this complementarity need not beperfect, in that there may be any number of base pair mismatches thatwill interfere with hybridization between the target sequence and thesingle stranded nucleic acids of the present invention. It is expectedthat the overall homology of the genes at the nucleotide level probablywill be about 40% or greater, probably about 60% or greater, and evenmore probably about 80% or greater; and in addition that there will becorresponding contiguous sequences of about 8-12 nucleotides or longer.However, if the number of mutations is so great that no hybridizationcan occur under even the least stringent of hybridization conditions,the sequence is not a complementary target sequence. Thus, by“substantially complementary” herein is meant that the probes aresufficiently complementary to the target sequences to hybridize undernormal reaction conditions, particularly high stringency conditions, asoutlined herein. Whether or not a sequence is unique to a CA geneaccording to this invention can be determined by techniques known tothose of skill in the art. For example, the sequence can be compared tosequences in databanks, e.g., GeneBank, to determine whether it ispresent in the uninfected host or other organisms. The sequence can alsobe compared to the known sequences of other viral agents, includingthose that are known to induce cancer.

A nucleic acid probe is generally single stranded but can be partlysingle and partly double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the oligonucleotide probes range from about 6, 8,10, 12, 15, 20, 30 to about 100 bases long, with from about 10 to about80 bases being preferred, and from about 30 to about 50 bases beingparticularly preferred. That is, generally entire genes are rarely usedas probes. In some embodiments, much longer nucleic acids can be used,up to hundreds of bases. The probes are sufficiently specific tohybridize to complementary template sequence under conditions known bythose of skill in the art. The number of mismatches between the probessequences and their complementary template (target) sequences to whichthey hybridize during hybridization generally do not exceed 15%, usuallydo not exceed 10% and preferably do not exceed 5%, as determined byFASTA (default settings).

Oligonucleotide probes can include the naturally-occurring heterocyclicbases normally found in nucleic acids (uracil, cytosine, thymine,adenine and guanine), as well as modified bases and base analogues. Anymodified base or base analogue compatible with hybridization of theprobe to a target sequence is useful in the practice of the invention.The sugar or glycoside portion of the probe can comprise deoxyribose,ribose, and/or modified forms of these sugars, such as, for example,2′-O-alkyl ribose. In a preferred embodiment, the sugar moiety is2′-deoxyribose; however, any sugar moiety that is compatible with theability of the probe to hybridize to a target sequence can be used.

In one embodiment, the nucleoside units of the probe are linked by aphosphodiester backbone, as is well known in the art. In additionalembodiments, internucleotide linkages can include any linkage known toone of skill in the art that is compatible with specific hybridizationof the probe including, but not limited to phosphorothioate,methylphosphonate, sulfamate (e.g., U.S. Pat. No. 5,470,967) andpolyamide (i.e., peptide nucleic acids). Peptide nucleic acids aredescribed in Nielsen et al. (1991) Science 254: 1497-1500, U.S. Pat. No.5,714,331, and Nielsen (1999) Curr. Opin. Biotechlnol. 10:71-75.

In certain embodiments, the probe can be a chimeric molecule; i.e., cancomprise more than one type of base or sugar subunit, and/or thelinkages can be of more than one type within the same primer. The probecan comprise a moiety to facilitate hybridization to its targetsequence, as are known in the art, for example, intercalators and/orminor groove binders. Variations of the bases, sugars, andinternucleoside backbone, as well as the presence of any pendant groupon the probe, will be compatible with the ability of the probe to bind,in a sequence-specific fashion, with its target sequence. A large numberof structural modifications, both known and to be developed, arepossible within these bounds. Advantageously, the probes according tothe present invention may have structural characteristics such that theyallow the signal amplification, such structural characteristics being,for example, branched DNA probes as those described by Urdea et al.(Nucleic Acids Symp. Ser., 24:197-200 (1991)) or in the European PatentNo. EP-0225,807. Moreover, synthetic methods for preparing the variousheterocyclic bases, sugars, nucleosides and nucleotides that form theprobe, and preparation of oligonucleotides of specific predeterminedsequence, are well-developed and known in the art. A preferred methodfor oligonucleotide synthesis incorporates the teaching of U.S. Pat. No.5,419,966.

Multiple probes may be designed for a particular target nucleic acid toaccount for polymorphism and/or secondary structure in the targetnucleic acid, redundancy of data and the like. In some embodiments,where more than one probe per sequence is used, either overlappingprobes or probes to different sections of a single target CA gene areused. That is, two, three, four or more probes, with three beingpreferred, are used to build in a redundancy for a particular target.The probes can be overlapping (i.e. have some sequence in common), orspecific for distinct sequences of a CA gene. When multiple targetpolynucleotides are to be detected according to the present invention,each probe or probe group corresponding to a particular targetpolynucleotide is situated in a discrete area of the microarray.

Probes may be in solution, such as in wells or on the surface of amicro-array, or attached to a solid support. Examples of solid supportmaterials that can be used include a plastic, a ceramic, a metal, aresin, a gel and a membrane. Useful types of solid supports includeplates, beads, magnetic material, microbeads, hybridization chips,membranes, crystals, ceramics and self-assembling monolayers. Apreferred embodiment comprises a two-dimensional or three-dimensionalmatrix, such as a gel or hybridization chip with multiple probe bindingsites (Pevzner et al., J. Biomol. Struc. & Dyn. 9:399-410, 1991; Maskosand Southern, Nuc. Acids Res. 20:1679-84, 1992). Hybridization chips canbe used to construct very large probe arrays that are subsequentlyhybridized with a target nucleic acid. Analysis of the hybridizationpattern of the chip can assist in the identification of the targetnucleotide sequence. Patterns can be manually or computer analyzed, butit is clear that positional sequencing by hybridization lends itself tocomputer analysis and automation. Algorithms and software, which havebeen developed for sequence reconstruction, are applicable to themethods described herein (R. Drmanac et al., J. Biomol. Struc. & Dyn.5:1085-1102, 1991; P. A. Pevzner, J. Biomol. Struc. & Dyn. 7:63-73,1989).

As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” herein is meant the association or binding between thenucleic acid probe and the solid support is sufficient to be stableunder the conditions of binding, washing, analysis, and removal asoutlined below. The binding can be covalent or non-covalent. By“non-covalent binding” and grammatical equivalents herein is meant oneor more of either electrostatic, hydrophilic, and hydrophobicinteractions. Included in non-covalent binding is the covalentattachment of a molecule, such as streptavidin, to the support and thenon-covalent binding of the biotinylated probe to the streptavidin. By“covalent binding” and grammatical equivalents herein is meant that thetwo moieties, the solid support and the probe, are attached by at leastone bond, including sigma bonds, pi bonds and coordination bonds.Covalent bonds can be formed directly between the probe and the solidsupport or can be formed by a cross linker or by inclusion of a specificreactive group on either the solid support or the probe or bothmolecules. Immobilization may also involve a combination of covalent andnon-covalent interactions.

Nucleic acid probes may be attached to the solid support by covalentbinding such as by conjugation with a coupling agent or by, covalent ornon-covalent binding such as electrostatic interactions, hydrogen bondsor antibody-antigen coupling, or by combinations thereof. Typicalcoupling agents include biotin/avidin, biotin/streptavidin,Staphylococcus aureus protein A/IgG antibody F_(c) fragment, andstreptavidin/protein A chimeras (T. Sano and C. R. Cantor,Bio/Technology 9:1378-81 (1991)), or derivatives or combinations ofthese agents. Nucleic acids may be attached to the solid support by aphotocleavable bond, an electrostatic bond, a disulfide bond, a peptidebond, a diester bond or a combination of these sorts of bonds. The arraymay also be attached to the solid support by a selectively releasablebond such as 4,4′-dimethoxytrityl or its derivative. Derivatives whichhave been found to be useful include 3 or4[bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or4[bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or4[bis-(4-methoxyphenyl)]-hydroxymethyl-benzoic acid, N-succinimidyl-3 or4[bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid, and salts of theseacids.

In general, the probes are attached to the biochip in a wide variety ofways, as will be appreciated by those in the art. As described herein,the nucleic acids can either be synthesized first, with subsequentattachment to the biochip, or can be directly synthesized on thebiochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. The solid phasesupport of the present invention can be of any solid materials andstructures suitable for supporting nucleotide hybridization andsynthesis. Preferably, the solid phase support comprises at least onesubstantially rigid surface on which the primers can be immobilized andthe reverse transcriptase reaction performed. The substrates with whichthe polynucleotide microarray elements are stably associated may befabricated from a variety of materials, including plastics, ceramics,metals, acrylamide, cellulose, nitrocellulose, glass, polystyrene,polyethylene vinyl acetate, polypropylene, polymethacrylate,polyethylene, polyethylene oxide, polysilicates, polycarbonates,Teflon®, fluorocarbons, nylon, silicon rubber, polyanhydrides,polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate,collagen, glycosaminoglycans, and polyamino acids. Substrates may betwo-dimensional or three-dimensional in form, such as gels, membranes,thin films, glasses, plates, cylinders, beads, magnetic beads, opticalfibers, woven fibers, etc. A preferred form of array is athree-dimensional array. A preferred three-dimensional array is acollection of tagged beads. Each tagged bead has different primersattached to it. Tags are detectable by signaling means such as color(Luminex, Illumina) and electromagnetic field (Pharmaseq) and signals ontagged beads can even be remotely detected (e.g., using optical fibers).The size of the solid support can be any of the standard microarraysizes, useful for DNA microarray technology, and the size may betailored to fit the particular machine being used to conduct a reactionof the invention. In general, the substrates allow optical detection anddo not appreciably fluoresce.

In a preferred embodiment, the surface of the biochip and the probe maybe derivatized with chemical functional groups for subsequent attachmentof the two. Thus, for example, the biochip is derivatized with achemical functional group including, but not limited to, amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups, for example using linkers as are known in the art; forexample, homo- or hetero-bifunctional linkers as are well known (see1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference). Inaddition, in some cases, additional linkers, such as alkyl groups(including substituted and heteroalkyl groups) may be used.

In this embodiment, the oligonucleotides are synthesized as is known inthe art, and then attached to the surface of the solid support. As willbe appreciated by those skilled in the art, either the 5′ or 3′ terminusmay be attached to the solid support, or attachment may be via aninternal nucleoside. In an additional embodiment, the immobilization tothe solid support may be very strong, yet non-covalent. For example,biotinylated oligonucleotides can be made, which bind to surfacescovalently coated with streptavidin, resulting in attachment.

The arrays may be produced according to any convenient methodology, suchas preforming the polynucleotide microarray elements and then stablyassociating them with the surface. Alternatively, the oligonucleotidesmay be synthesized on the surface, as is known in the art. A number ofdifferent array configurations and methods for their production areknown to those of skill in the art and disclosed in WO 95/25116 and WO95/35505 (photolithographic techniques), U.S. Pat. No. 5,445,934 (insitu synthesis by photolithography), U.S. Pat. No. 5,384,261 (in situsynthesis by mechanically directed flow paths); and U.S. Pat. No.5,700,637 (synthesis by spotting, printing or coupling); the disclosureof which are herein incorporated in their entirety by reference. Anothermethod for coupling DNA to beads uses specific ligands attached to theend of the DNA to link to ligand-binding molecules attached to a bead.Possible ligand-binding partner pairs includebiotin-avidin/streptavidin, or various antibody/antigen pairs such asdigoxygenin-antidigoxygenin antibody (Smith et al., “Direct MechanicalMeasurements of the Elasticity of Single DNA Molecules by Using MagneticBeads,” Science 258:1122-1126 (1992)). Covalent chemical attachment ofDNA to the support can be accomplished by using standard coupling agentsto link the 5′-phosphate on the DNA to coated microspheres through aphosphoamidate bond. Methods for immobilization of oligonucleotides tosolid-state substrates are well established. See Pease et al., Proc.Natl. Acad. Sci. USA 91(11):5022-5026 (1994). A preferred method ofattaching oligonucleotides to solid-state substrates is described by Guoet al., Nucleic Acids Res. 22:5456-5465 (1994). Immobilization can beaccomplished either by in situ DNA synthesis (Maskos and Southern,Nucleic Acids Research, 20:1679-1684 (1992) or by covalent attachment ofchemically synthesized oligonucleotides (Guo et al., supra) incombination with robotic arraying technologies.

In addition to the solid-phase technology represented by biochip arrays,gene expression can also be quantified using liquid-phase arrays. Onesuch system is kinetic polymerase chain reaction (PCR). Kinetic PCRallows for the simultaneous amplification and quantification of specificnucleic acid sequences. The specificity is derived from syntheticoligonucleotide primers designed to preferentially adhere tosingle-stranded nucleic acid sequences bracketing the target site. Thispair of oligonucleotide primers form specific, non-covalently boundcomplexes on each strand of the target sequence. These complexesfacilitate in vitro transcription of double-stranded DNA in oppositeorientations. Temperature cycling of the reaction mixture creates acontinuous cycle of primer binding, transcription, and re-melting of thenucleic acid to individual strands. The result is an exponentialincrease of the target dsDNA product. This product can be quantified inreal time either through the use of an intercalating dye or a sequencespecific probe. SYBR® Greene I, is an example of an intercalating dye,that preferentially binds to dsDNA resulting in a concomitant increasein the fluorescent signal. Sequence specific probes, such as used withTaqMan® technology, consist of a fluorochrome and a quenching moleculecovalently bound to opposite ends of an oligonucleotide. The probe isdesigned to selectively bind the target DNA sequence between the twoprimers. When the DNA strands are synthesized during the PCR reaction,the fluorochrome is cleaved from the probe by the exonuclease activityof the polymerase resulting in signal dequenching. The probe signalingmethod can be more specific than the intercalating dye method, but ineach case, signal strength is proportional to the dsDNA productproduced. Each type of quantification method can be used in multi-wellliquid phase arrays with each well representing primers and/or probesspecific to nucleic acid sequences of interest. When used with messengerRNA preparations of tissues or cell lines, an array of probe/primerreactions can simultaneously quantify the expression of multiple geneproducts of interest. See Germer, S., et al., Genome Res. 10:258-266(2000); Heid, C. A., et al., Genome Res. 6, 986-994 (1996).

Expression of CA Proteins

In a preferred embodiment, CA nucleic acids encoding CA proteins areused to make a variety of expression vectors to express CA proteinswhich can then be used in screening assays, as described below. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the CAprotein. The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the CA protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the CA protein in Bacillus. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences that flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

The CA proteins of the present invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding a CA protein, under the appropriate conditions to induce orcause expression of the CA protein. The conditions appropriate for CAprotein expression will vary with the choice of the expression vectorand the host cell, and will be easily ascertained by one skilled in theart through routine experimentation. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect, plant and animal cells, including mammalian cells. Ofparticular interest are Drosophila melanogaster cells, Saccharomycescerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line(a macrophage cell line) and human cells and cell lines.

In a preferred embodiment, the CA proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A preferred expression vector system is aretroviral vector system such as is generally described inPCT/US97/01019 and PCT/US97/01048, both of which are hereby expresslyincorporated by reference. Of particular use as mammalian promoters arethe promoters from mammalian viral genes, since the viral genes areoften highly expressed and have a broad host range. Examples include theSV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirusmajor late promoter, herpes simplex virus promoter, and the CMVpromoter. Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. Examples of transcriptionterminator and polyadenylation signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, are well known in the art, and will vary withthe host cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, CA proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. In addition toa functioning promoter sequence, an efficient ribosome binding site isdesirable. The expression vector may also include a signal peptidesequence that provides for secretion of the CA protein in bacteria. Theprotein is either secreted into the growth media (gram-positivebacteria) or into the periplasmic space, located between the inner andouter membrane of the cell (gram-negative bacteria). The bacterialexpression vector may also include a selectable marker gene to allow forthe selection of bacterial strains that have been transformed. Suitableselection genes include genes that render the bacteria resistant todrugs such as ampicillin, chloramphenicol, erythromycin, kanamycin,neomycin and tetracycline. Selectable markers also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways. These components are assembled into expressionvectors. Expression vectors for bacteria are well known in the art, andinclude vectors for Bacillus subtilis, E. coli, Streptococcus cremoris,and Streptococcus lividans, among others. The bacterial expressionvectors are transformed into bacterial host cells using techniques wellknown in the art, such as calcium chloride treatment, electroporation,and others.

In one embodiment, CA proteins are produced in insect cells. Expressionvectors for the transformation of insect cells, and in particular,baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, CA protein is produced in yeast cells. Yeastexpression systems are well known in the art, and include expressionvectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichiaguillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowialipolytica.

The CA protein may also be made as a fusion protein, using techniqueswell known in the art. Thus, for example, for the creation of monoclonalantibodies. If the desired epitope is small, the CA protein may be fusedto a carrier protein to form an immunogen. Alternatively, the CA proteinmay be made as a fusion protein to increase expression, or for otherreasons. For example, when the CA protein is a CA peptide, the nucleicacid encoding the peptide may be linked to other nucleic acid forexpression purposes.

In one embodiment, the CA nucleic acids, proteins and antibodies of theinvention are labeled. By “labeled” herein is meant that a compound hasat least one element, isotope or chemical compound attached to enablethe detection of the compound. In general, labels fall into threeclasses: a) isotopic labels, which may be radioactive or heavy isotopes;b) immune labels, which may be antibodies or antigens; and c) colored orfluorescent dyes. The labels may be incorporated into the CA nucleicacids, proteins and antibodies at any position. For example, the labelshould be capable of producing, either directly or indirectly, adetectable signal. The detectable moiety may be a radioisotope, such as³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescent compound,such as fluorescein isothiocyanate, rhodamine, or luciferin, or anenzyme, such as alkaline phosphatase, beta-galactosidase or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe label may be employed, including those methods described by Hunteret al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014(1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J.Histochem. and Cytochem., 30:407 (1982).

Accordingly, the present invention also provides CA protein sequences. ACA protein of the present invention may be identified in several ways.“Protein” in this sense includes proteins, polypeptides, and peptides.As will be appreciated by those in the art, the nucleic acid sequencesof the invention can be used to generate protein sequences. There are avariety of ways to do this, including cloning the entire gene andverifying its frame and amino acid sequence, or by comparing it to knownsequences to search for homology to provide a frame, assuming the CAprotein has homology to some protein in the database being used.Generally, the nucleic acid sequences are input into a program that willsearch all three frames for homology. This is done in a preferredembodiment using the following NCBI Advanced BLAST parameters. Theprogram is blastx or blastn. The database is nr. The input data is as“Sequence in FASTA format”. The organism list is “none”. The “expect” is10; the filter is default. The “descriptions” is 500, the “alignments”is 500, and the “alignment view” is pairwise. The “query Genetic Codes”is standard (1). The matrix is BLOSUM 62; gap existence cost is 11, perresidue gap cost is 1; and the lambda ratio is 0.85 default. Thisresults in the generation of a putative protein sequence.

In general, the term “polypeptide” as used herein refers to both thefull-length polypeptide encoded by the recited polynucleotide, thepolypeptide encoded by the gene represented by the recitedpolynucleotide, as well as portions or fragments thereof. The presentinvention encompasses variants of the naturally occurring proteins,wherein such variants are homologous or substantially similar to thenaturally occurring protein, and can be of an origin of the same ordifferent species as the naturally occurring protein (e.g., human,murine, or some other species that naturally expresses the recitedpolypeptide, usually a mammalian species). In general, variantpolypeptides have a sequence that has at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, usually at least about 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% and more usually at least about 99% sequenceidentity with a differentially expressed polypeptide described herein,as determined by the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489. The variant polypeptides can be naturally or non-naturallyglycosylated, i.e., the polypeptide has a glycosylation pattern thatdiffers from the glycosylation pattern found in the correspondingnaturally occurring protein.

Also within the scope of the invention are variants. Variants ofpolypeptides include mutants, fragments, and fusions. Mutants caninclude amino acid substitutions, additions or deletions. The amino acidsubstitutions can be conservative amino acid substitutions orsubstitutions to eliminate non-essential amino acids, such as to alter aglycosylation site, a phosphorylation site or an acetylation site, or tominimize misfolding by substitution or deletion of one or more cysteineresidues that are not necessary for function. Conservative amino acidsubstitutions are those that preserve the general charge,hydrophobicity/hydrophilicity, and/or steric bulk of the amino acidsubstituted. Variants can be designed so as to retain or have enhancedbiological activity of a particular region of the protein (e.g., afunctional domain and/or, where the polypeptide is a member of a proteinfamily, a region associated with a consensus sequence). Selection ofamino acid alterations for production of variants can be based upon theaccessibility (interior vs. exterior) of the amino acid (see, e.g., Goet al, Int. J. Peptide Protein Res. (1980) 15:211), the thermostabilityof the variant polypeptide (see, e.g., Querol et al., Prot. Eng. (1996)9:265), desired glycosylation sites (see, e.g., Olsen and Thomsen, J.Gen. Microbiol. (1991) 137:579), desired disulfide bridges (see, e.g.,Clarke et al., Biochemistry (1993) 32:4322; and Wakarchuk et al.,Protein Eng. (1994) 7:1379), desired metal binding sites (see, e.g.,Toma et al., Biochemistry (1991) 30:97, and Haezerbrouck et al., ProteinEng. (1993) 6:643), and desired substitutions within proline loops (see,e.g., Masul et al., Appl. Env. Microbiol. (1994) 60:3579).Cysteine-depleted muteins can be produced as disclosed in U.S. Pat. No.4,959,314.

Variants also include fragments of the polypeptides disclosed herein,particularly biologically active fragments and/or fragmentscorresponding to functional domains. Fragments of interest willtypically be at least about 8 amino acids (aa) 10 aa, 15 aa, 20 aa, 25aa, 30 aa, 35 aa, 40 aa, to at least about 45 aa in length, usually atleast about 50 aa in length, at least about 75 aa, at least about 100aa, at least about 125 aa, at least about 150 aa in length, at leastabout 200 aa, at least about 300 aa, at least about 400 aa and can be aslong as 500 aa in length or longer, but will usually not exceed about1000 aa in length, where the fragment will have a stretch of amino acidsthat is identical to a polypeptide encoded by a polynucleotide having asequence of any one of the polynucleotide sequences provided herein, ora homolog thereof. The protein variants described herein are encoded bypolynucleotides that are within the scope of the invention. The geneticcode can be used to select the appropriate codons to construct thecorresponding variants.

While altered expression of the polynucleotides associated with canceris observed, altered levels of expression of the polypeptides encoded bythese polynucleotides may likely play a role in cancers.

Also included within one embodiment of CA proteins are amino acidvariants of the naturally occurring sequences, as determined herein.Preferably, the variants are preferably greater than about 75%homologous to the wild-type sequence, more preferably greater than about80%, even more preferably greater than about 85% and most preferablygreater than 90%. The present application is also directed to proteinscontaining polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to a CA polypeptide sequence set forth herein. As fornucleic acids, homology in this context means sequence similarity oridentity, with identity being preferred. This homology will bedetermined using standard techniques known in the art as are outlinedabove for the nucleic acid homologies.

CA proteins of the present invention may be shorter or longer than thewild type amino acid sequences. Thus, in a preferred embodiment,included within the definition of CA proteins are portions or fragmentsof the wild type sequences herein. In addition, as outlined above, theCA nucleic acids of the invention may be used to obtain additionalcoding regions, and thus additional protein sequence, using techniquesknown in the art.

In a preferred embodiment, the CA proteins are derivative or variant CAproteins as compared to the wild-type sequence. That is, as outlinedmore fully below, the derivative CA peptide will contain at least oneamino acid substitution, deletion or insertion, with amino acidsubstitutions being particularly preferred. The amino acid substitution,insertion or deletion may occur at any residue within the CA peptide.

Also included in an embodiment of CA proteins of the present inventionare amino acid sequence variants. These variants fall into one or moreof three classes: substitutional, insertional or deletional variants.These variants ordinarily are prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the CA protein, using cassette or PCRmutagenesis or other techniques well known in the art, to produce DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture as outlined above. However, variant CA protein fragmentshaving up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. Amino acid sequence variants arecharacterized by the predetermined nature of the variation, a featurethat sets them apart from naturally occurring allelic or interspeciesvariation of the CA protein amino acid sequence. The variants typicallyexhibit the same qualitative biological activity as the naturallyoccurring analogue, although variants can also be selected which havemodified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed CA variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and LARmutagenesis. Screening of the mutants is done using assays of CA proteinactivities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the CA protein are desired,substitutions are generally made in accordance with the following chart:

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made full length to moresignificantly affect one or more of the following: the structure of thepolypeptide backbone in the area of the alteration (e.g., thealpha-helical or beta-sheet structure); the charge or hydrophobicity ofthe molecule at the target site; and the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the polypeptide's properties are those in which (a) ahydrophilic residue, e.g. seryl or threonyl is substituted for (or by) ahydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g. lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky sidechain, e.g. phenylalanine, is substituted for (or by) one not having aside chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the CA proteins as needed. Alternatively, the variantmay be designed such that the biological activity of the CA protein isaltered. For example, glycosylation sites may be altered or removed,dominant negative mutations created, etc.

Covalent modifications of CA polypeptides are included within the scopeof this invention, for example for use in screening. One type ofcovalent modification includes reacting targeted amino acid residues ofa CA polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of aCA polypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking CA polypeptides to a water-insoluble supportmatrix or surface for use in the method for purifying anti-CA antibodiesor screening assays, as is more fully described below. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl, threonyl or tyrosyl residues, methylation ofthe a-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the CA polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence CApolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence CA polypeptide.

Addition of glycosylation sites to CA polypeptides may be accomplishedby altering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence CA polypeptide (forO-linked glycosylation sites). The CA amino acid sequence may optionallybe altered through changes at the DNA level, particularly by mutatingthe DNA encoding the CA polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theCA polypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are described in the art, e.g., in WO 87/05330published 11 Sep. 1987, and in Aplin and Wriston, LA Crit. Rev.Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the CA polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of CA comprises linking the CApolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

CA polypeptides of the present invention may also be modified in a wayto form chimeric molecules comprising a CA polypeptide fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of a CA polypeptide with a tagpolypeptide that provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the CA polypeptide, although internal fusions mayalso be tolerated in some instances. The presence of such epitope-taggedforms of a CA polypeptide can be detected using an antibody against thetag polypeptide. Also, provision of the epitope tag enables the CApolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a CA polypeptide with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

Also included with the definition of CA protein in one embodiment areother CA proteins of the CA family, and CA proteins from otherorganisms, which are cloned and expressed as outlined below. Thus, probeor degenerate polymerase chain reaction (PCR) primer sequences may beused to find other related CA proteins from humans or other organisms.As will be appreciated by those in the art, particularly useful probeand/or PCR primer sequences include the unique areas of the CA nucleicacid sequence. As is generally known in the art, preferred PCR primersare from about 15 to about 35 nucleotides in length, with from about 20to about 30 being preferred, and may contain inosine as needed. Theconditions for the PCR reaction are well known in the art.

In addition, as is outlined herein, CA proteins can be made that arelonger than those encoded by the nucleic acids of the figures, forexample, by the elucidation of additional sequences, the addition ofepitope or purification tags, the addition of other fusion sequences,etc.

CA proteins may also be identified as being encoded by CA nucleic acids.Thus, CA proteins are encoded by nucleic acids that will hybridize tothe sequences of the sequence listings, or their complements, asoutlined herein.

CA Antigens and Antibodies Thereto

In one embodiment, the invention provides CA specific antibodies. In apreferred embodiment, when the CA protein is to be used to generateantibodies, for example for immunotherapy, the CA protein should shareat least one epitope or determinant with the full-length protein. By“epitope” or “determinant” herein is meant a portion of a protein thatwill generate and/or bind an antibody or T-cell receptor in the contextof MHC. Thus, in most instances, antibodies made to a smaller CA proteinwill be able to bind to the full-length protein. In a preferredembodiment, the epitope is unique; that is, antibodies generated to aunique epitope show little or no cross-reactivity.

Any polypeptide sequence encoded by the CA polynucleotide sequences maybe analyzed to determine certain preferred regions of the polypeptide.Regions of high antigenicity are determined from data by DNASTARanalysis by choosing values that represent regions of the polypeptidethat are likely to be exposed on the surface of the polypeptide in anenvironment in which antigen recognition may occur in the process ofinitiation of an immune response. For example, the amino acid sequenceof a polypeptide encoded by a CA polynucleotide sequence may be analyzedusing the default parameters of the DNASTAR computer algorithm (DNASTAR,Inc., Madison, Wis.; http://www.dnastar.com/).

Polypeptide features that may be routinely obtained using the DNASTARcomputer algorithm include, but are not limited to, Garnier-Robsonalpha-regions, beta-regions, turn-regions, and coil-regions (Garnier etal. J. Mol. Biol., 120: 97 (1978)); Chou-Fasman alpha-regions,beta-regions, and turn-regions (Adv. in Enzymol., 47:45-148 (1978));Kyte-Doolittle hydrophilic regions and hydrophobic regions (J. Mol.Biol., 157:105-132 (1982)); Eisenberg alpha- and beta-amphipathicregions; Karplus-Schulz flexible regions; Emini surface-forming regions(J. Virol., 55(3):836-839 (1985)); and Jameson-Wolf regions of highantigenic index (CABIOS, 4(1):181-186 (1988)). Kyte-Doolittlehydrophilic regions and hydrophobic regions, Emini surface-formingregions, and Jameson-Wolf regions of high antigenic index (i.e.,containing four or more contiguous amino acids having an antigenic indexof greater than or equal to 1.5, as identified using the defaultparameters of the Jameson-Wolf program) can routinely be used todetermine polypeptide regions that exhibit a high degree of potentialfor antigenicity. One approach for preparing antibodies to a protein isthe selection and preparation of an amino acid sequence of all or partof the protein, chemically synthesizing the sequence and injecting itinto an appropriate animal, typically a rabbit, hamster or a mouse.Oligopeptides can be selected as candidates for the production of anantibody to the CA protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein. Additional oligopeptides can be determined using, for example,the Antigenicity Index, Welling, G. W. et al., FEBS Lett. 188:215-218(1985), incorporated herein by reference.

In one embodiment, the term “antibody” includes antibody fragments, asare known in the art, including Fab, Fab₂, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include a protein encoded by anucleic acid of the figures or fragment thereof or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

The antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The immunizing agent will typically include a polypeptide encodedby a nucleic acid of Tables 1-16, or fragment thereof or a fusionprotein thereof. Generally, either peripheral blood lymphocytes (“PBLs”)are used if cells of human origin are desired, or spleen cells or lymphnode cells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Monoclonal antibody technology is used in implementing research,diagnosis and therapy. Monoclonal antibodies are used inradioimmunoassays, enzyme-linked immunosorbent assays,immunocytopathology, and flow cytometry for in vitro diagnosis, and invivo for diagnosis and immunotherapy of human disease. Waldmann, T. A.(1991) Science 252:1657-1662. In particular, monoclonal antibodies havebeen widely applied to the diagnosis and therapy of cancer, wherein itis desirable to target malignant lesions while avoiding normal tissue.See, e.g., U.S. Pat. Nos. 4,753,894 to Frankel, et al.; 4,938,948 toRing et al.; and 4,956,453 to Bjorn et al.

In one embodiment, the antibodies are bispecific antibodies. Bispecificantibodies are monoclonal, preferably human or humanized, antibodiesthat have binding specificities for at least two different antigens. Anumber of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220 -4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients. In the present case, one of thebinding specificities is for a protein encoded by a nucleic acid ofTables 1-16, or a fragment thereof, the other one is for any otherantigen, and preferably for a cell-surface protein or receptor orreceptor subunit, preferably one that is tumor specific.

In a preferred embodiment, the antibodies to CA are capable of reducingor eliminating the biological function of CA, as is described below.That is, the addition of anti-CA antibodies (either polyclonal orpreferably monoclonal) to CA (or cells containing CA) may reduce oreliminate the CA activity. Generally, at least a 25% decrease inactivity is preferred, with at least about 50% being particularlypreferred and about a 95-100% decrease being especially preferred.

In a preferred embodiment the antibodies to the CA proteins arehumanized antibodies. “Humanized” antibodies refer to a molecule havingan antigen binding site that is substantially derived from animmunoglobulin from a non-human species and the remaining immunoglobulinstructure of the molecule based upon the structure and/or sequence of ahuman immunoglobulin. The antigen binding site may comprise eithercomplete variable domains fused onto constant domains or only thecomplementarity determining regions (CDRs) grafted onto appropriateframework regions in the variable domains. Antigen binding sites may bewild type or modified by one or more amino acid substitutions, e.g.,modified to resemble human immunoglobulin more closely. Alternatively, ahumanized antibody may be derived from a chimeric antibody that retainsor substantially retains the antigen-binding properties of the parental,non-human, antibody but which exhibits diminished immunogenicity ascompared to the parental antibody when administered to humans. Thephrase “chimeric antibody,” as used herein, refers to an antibodycontaining sequence derived from two different antibodies (see, e.g.,U.S. Pat. No. 4,816,567) that typically originate from differentspecies. Typically, in these chimeric antibodies, the variable region ofboth light and heavy chains mimics the variable regions of antibodiesderived from one species of mammals, while the constant portions arehomologous to the sequences in antibodies derived from another. Mosttypically, chimeric antibodies comprise human and murine antibodyfragments, generally human constant and mouse variable regions.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework residues (FR) regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin (Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)). One clear advantage to suchchimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

Because humanized antibodies are far less immunogenic in humans than theparental mouse monoclonal antibodies, they can be used for the treatmentof humans with far less risk of anaphylaxis. Thus, these antibodies maybe preferred in therapeutic applications that involve in vivoadministration to a human such as, e.g., use as radiation sensitizersfor the treatment of neoplastic disease or use in methods to reduce theside effects of, e.g., cancer therapy. Methods for humanizing non-humanantibodies are well known in the art. Generally, a humanized antibodyhas one or more amino acid residues introduced into it from a sourcethat is non-human. These non-human amino acid residues are oftenreferred to as import residues, which are typically taken from an importvariable domain. Humanization can be essentially performed following themethod of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies [Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Humanizedantibodies may be achieved by a variety of methods including, forexample: (1) grafting the non-human complementarity determining regions(CDRs) onto a human framework and constant region (a process referred toin the art as “humanizing”), or, alternatively, (2) transplanting theentire non-human variable domains, but “cloaking” them with a human-likesurface by replacement of surface residues (a process referred to in theart as “veneering”). In the present invention, humanized antibodies willinclude both “humanized” and “veneered” antibodies. Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); Joneset al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad.Sci, U.S.A., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol.,44:65-92 (1988); Verhoeyer et al., Science 239:1534-1536 (1988); Padlan,Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217(1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991)each of which is incorporated herein by reference.

The phrase “complementarity determining region” refers to amino acidsequences which together define the binding affinity and specificity ofthe natural Fv region of a native immunoglobulin binding site. See,e.g., Chothia et al., J. Mol. Biol. 196:901-917 (1987); Kabat et al.,U.S. Dept. of Health and Human Services NIH Publication No. 91-3242(1991). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In the presentinvention, mouse constant regions are substituted by human constantregions. The constant regions of the subject humanized antibodies arederived from human immunoglobulins. The heavy chain constant region canbe selected from any of the five isotypes: alpha, delta, epsilon, gammaor mu. One method of humanizing antibodies comprises aligning thenon-human heavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region that disturb the structure of theCDRs until the predicted conformation of the humanized sequence modelclosely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g, via Ashwellreceptors. See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 which areincorporated herein by reference.

Humanized antibodies to CA polypeptides can also be produced usingtransgenic animals that are engineered to contain human immunoglobulinloci. For example, WO 98/24893 discloses transgenic animals having ahuman Ig locus wherein the animals do not produce functional endogenousimmunoglobulins due to the inactivation of endogenous heavy and lightchain loci. WO 91/10741 also discloses transgenic non-primate mammalianhosts capable of mounting an immune response to an immunogen, whereinthe antibodies have primate constant and/or variable regions, andwherein the endogenous immunoglobulin-encoding loci are substituted orinactivated. WO 96/30498 discloses the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody-producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. The monoclonalantibodies can be tested for the ability to inhibit or neutralize thebiological activity or physiological effect of the correspondingprotein.

In the present invention, CA polypeptides of the invention and variantsthereof are used to immunize a transgenic animal as described above.Monoclonal antibodies are made using methods known in the art, and thespecificity of the antibodies is tested using isolated CA polypeptides.Methods for preparation of the human or primate CA or an epitope thereofinclude, but are not limited to chemical synthesis, recombinant DNAtechniques or isolation from biological samples. Chemical synthesis of apeptide can be performed, for example, by the classical Merrifeld methodof solid phase peptide synthesis (Merrifeld, J. Am. Chem. Soc. 85:2149,1963 which is incorporated by reference) or the FMOC strategy on a RapidAutomated Multiple Peptide Synthesis system (E.I. du Pont de NemoursCompany, Wilmington, Del.) (Caprino and Han, J. Org Chem. 37:3404, 1972which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified CA proteins usually by ELISA or by bioassay based upon theability to block the action of CA proteins. When using avian species,e.g., chicken, turkey and the like, the antibody can be isolated fromthe yolk of the egg. Monoclonal antibodies can be prepared after themethod of Milstein and Kohler by fusing splenocytes from immunized micewith continuously replicating tumor cells such as myeloma or lymphomacells. (Milstein and Kohler, Nature 256:495-497, 1975; Gulfre andMilstein, Methods in Enzymology: Immunochemical Techniques 73:1-46,Langone and Banatis eds., Academic Press, 1981 which are incorporated byreference). The hybridoma cells so formed are then cloned by limitingdilution methods and supernates assayed for antibody production byELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an overexpression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving overexpression ofa CA polypeptide by treatment of a patient with specific antibodies tothe CA protein.

Specific antibodies, either polyclonal or monoclonal, to the CA proteinscan be produced by any suitable method known in the art as discussedabove. For example, murine or human monoclonal antibodies can beproduced by hybridoma technology or, alternatively, the CA proteins, oran immunologically active fragment thereof, or an anti-idiotypicantibody, or fragment thereof can be administered to an animal to elicitthe production of antibodies capable of recognizing and binding to theCA proteins. Such antibodies can be from any class of antibodiesincluding, but not limited to IgG, IgA, IgM, IgD, and IgE or in the caseof avian species, IgY and from any subclass of antibodies.

By immunotherapy is meant treatment of a cancer with an antibody raisedagainst a CA protein. As used herein, immunotherapy can be passive oractive. Passive immunotherapy as defined herein is the passive transferof antibody to a recipient (patient). Active immunization is theinduction of antibody and/or T-cell responses in a recipient (patient).Induction of an immune response is the result of providing the recipientwith an antigen to which antibodies are raised. As appreciated by one ofordinary skill in the art, the antigen may be provided by injecting apolypeptide against which antibodies are desired to be raised into arecipient, or contacting the recipient with a nucleic acid capable ofexpressing the antigen and under conditions for expression of theantigen.

In a preferred embodiment, oncogenes which encode secreted growthfactors may be inhibited by raising antibodies against CA proteins thatare secreted proteins as described above. Without being bound by theory,antibodies used for treatment, bind and prevent the secreted proteinfrom binding to its receptor, thereby inactivating the secreted CAprotein.

In another preferred embodiment, the CA protein to which antibodies areraised is a transmembrane protein. Without being bound by theory,antibodies used for treatment, bind the extracellular domain of the CAprotein and prevent it from binding to other proteins, such ascirculating ligands or cell-associated molecules. The antibody may causedown-regulation of the transmembrane CA protein. As will be appreciatedby one of ordinary skill in the art, the antibody may be a competitive,non-competitive or uncompetitive inhibitor of protein binding to theextracellular domain of the CA protein. The antibody is also anantagonist of the CA protein. Further, the antibody prevents activationof the transmembrane CA protein. In one aspect, when the antibodyprevents the binding of other molecules to the CA protein, the antibodyprevents growth of the cell. The antibody may also sensitize the cell tocytotoxic agents, including, but not limited to TNF-α, TNF-β, IL-1,INF-γ and IL-2, or chemotherapeutic agents including 5FU, vinblastine,actinomycin D, cisplatin, methotrexate, and the like. In some instancesthe antibody belongs to a sub-type that activates serum complement whencomplexed with the transmembrane protein thereby mediating cytotoxicity.Thus, cancers may be treated by administering to a patient antibodiesdirected against the transmembrane CA protein.

In another preferred embodiment, the antibody is conjugated to atherapeutic moiety. In one aspect the therapeutic moiety is a smallmolecule that modulates the activity of the CA protein. In anotheraspect the therapeutic moiety modulates the activity of moleculesassociated with or in close proximity to the CA protein. The therapeuticmoiety may inhibit enzymatic activity such as protease or protein kinaseactivity associated with cancer.

In a preferred embodiment, the therapeutic moiety may also be acytotoxic agent. In this method, radioisotopes, natural toxins,chemotherapy agents, or other substances (such as biological responsemodifiers) are chemically linked or conjugated to a monoclonal antibodyto form “immunoconjugates” and “immunotoxins” which target the cytotoxicagent to tumor tissue or cells resulting in a reduction in the number ofafflicted cells, thereby reducing symptoms associated with cancers,including lymphoma. Cytotoxic agents are numerous and varied andinclude, but are not limited to, cytotoxic drugs or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diphtheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies raised against CA proteins, or binding of aradionuclide to a chelating agent that has been covalently attached tothe antibody. Targeting the therapeutic moiety to transmembrane CAproteins not only serves to increase the local concentration oftherapeutic moiety in the cancer of interest, i.e., lymphoma, but alsoserves to reduce deleterious side effects that may be associated withthe therapeutic moiety. A number of investigators have used monoclonalantibodies as carriers of cytotoxic substances in attempts toselectively direct those agents to malignant tissue. More particularly,a number of monoclonal antibodies have been conjugated to toxins such asricin, abrin, diphtheria toxin and Pseudomonas exotoxin or toenzymatically active portions (A chains) thereof via heterobifunctionalagents. See, e.g., U.S. Pat. No. 4,753,894 to Frankel et al.; Nevelle,et al. (1982) Immunol Rev 62:75-91; Ross et al. (1980) Eur. J. Biochem104; Vitteta et al. (1982) Immunol Rev 62:158-183; Raso et al. (1982)Cancer Res 42:457-464, and Trowbridge et al. (1981) Nature 294:171-173.

In another preferred embodiment, the CA protein against which theantibodies are raised is an intracellular protein. In this case, theantibody may be conjugated to a protein that facilitates entry into thecell. In one case, the antibody enters the cell by endocytosis. Inanother embodiment, a nucleic acid encoding the antibody is administeredto the individual or cell. Moreover, wherein the CA protein can betargeted within a cell, e.g., the nucleus, an antibody thereto containsa signal for that target localization, e.g., a nuclear localizationsignal.

The CA antibodies of the invention specifically bind to CA proteins. By“specifically bind” herein is meant that the antibodies bind to theprotein with a binding constant in the range of 10⁻⁴-10⁻⁶ M⁻¹, with apreferred range being 10⁻⁷-10⁻⁹ M⁻⁴.

In a preferred embodiment, the CA protein is purified or isolated afterexpression. CA proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the CA protein may be purified using a standard anti-CA antibody column.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. For general guidance in suitablepurification techniques, see Scopes, R., Protein Purification,Springer-Verlag, NY (1982). The degree of purification necessary willvary depending on the use of the CA protein. In some instances nopurification will be necessary.

Detection of Cancer Phenotype

Once expressed and purified if necessary, the CA proteins and nucleicacids are useful in a number of applications. In one aspect, theexpression levels of genes are determined for different cellular statesin the cancer phenotype; that is, the expression levels of genes innormal tissue and in cancer tissue (and in some cases, for varyingseverities of lymphoma that relate to prognosis, as outlined below) areevaluated to provide expression profiles. An expression profile of aparticular cell state or point of development is essentially a“fingerprint” of the state; while two states may have any particulargene similarly expressed, the evaluation of a number of genessimultaneously allows the generation of a gene expression profile thatis unique to the state of the cell. By comparing expression profiles ofcells in different states, information regarding which genes areimportant (including both up- and down-regulation of genes) in each ofthese states is obtained. Then, diagnosis may be done or confirmed: doestissue from a particular patient have the gene expression profile ofnormal or cancer tissue.

“Differential expression,” or equivalents used herein, refers to bothqualitative as well as quantitative differences in the temporal and/orcellular expression patterns of genes, within and among the cells. Thus,a differentially expressed gene can qualitatively have its expressionaltered, including an activation or inactivation, in, for example,normal versus cancer tissue. That is, genes may be turned on or turnedoff in a particular state, relative to another state. As is apparent tothe skilled artisan, any comparison of two or more states can be made.Such a qualitatively regulated gene will exhibit an expression patternwithin a state or cell type which is detectable by standard techniquesin one such state or cell type, but is not detectable in both.Alternatively, the determination is quantitative in that expression isincreased or decreased; that is, the expression of the gene is eitherup-regulated, resulting in an increased amount of transcript, ordown-regulated, resulting in a decreased amount of transcript. Thedegree to which expression differs need only be large enough to quantifyvia standard characterization techniques as outlined below, such as byuse of Affymetrix GeneChip® expression arrays, Lockhart, NatureBiotechnology, 14:1675-1680 (1996), hereby expressly incorporated byreference. Other techniques include, but are not limited to,quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection. As outlined above, preferably the change in expression (i.e.upregulation or downregulation) is at least about 50%, more preferablyat least about 100%, more preferably at least about 150%, morepreferably, at least about 200%, with from 300 to at least 1000% beingespecially preferred.

As will be appreciated by those in the art, this may be done byevaluation at either the gene transcript, or the protein level; that is,the amount of gene expression may be monitored using nucleic acid probesto the DNA or RNA equivalent of the gene transcript, and thequantification of gene expression levels, or, alternatively, the finalgene product itself (protein) can be monitored, for example through theuse of antibodies to the CA protein and standard immunoassays (ELISAs,etc.) or other techniques, including mass spectroscopy assays, 2D gelelectrophoresis assays, etc. Thus, the proteins corresponding to CAgenes, i.e. those identified as being important in a particular cancerphenotype, i.e., lymphoma, can be evaluated in a diagnostic testspecific for that cancer.

In a preferred embodiment, gene expression monitoring is done and anumber of genes, i.e. an expression profile, is monitoredsimultaneously, although multiple protein expression monitoring can bedone as well. Similarly, these assays may be done on an individual basisas well.

In this embodiment, the CA nucleic acid probes may be attached tobiochips as outlined herein for the detection and quantification of CAsequences in a particular cell. The assays are done as is known in theart. As will be appreciated by those in the art, any number of differentCA sequences may be used as probes, with single sequence assays beingused in some cases, and a plurality of the sequences described hereinbeing used in other embodiments. In addition, while solid-phase assaysare described, any number of solution based assays may be done as well.

In a preferred embodiment, both solid and solution based assays may beused to detect CA sequences that are up-regulated or down-regulated incancers as compared to normal tissue. In instances where the CA sequencehas been altered but shows the same expression profile or an alteredexpression profile, the protein will be detected as outlined herein.

In a preferred embodiment nucleic acids encoding the CA protein aredetected. Although DNA or RNA encoding the CA protein may be detected,of particular interest are methods wherein the mRNA encoding a CAprotein is detected. The presence of mRNA in a sample is an indicationthat the CA gene has been transcribed to form the mRNA, and suggeststhat the protein is expressed. Probes to detect the mRNA can be anynucleotide/deoxynucleotide probe that is complementary to and base pairswith the mRNA and includes but is not limited to oligonucleotides, cDNAor RNA. Probes also should contain a detectable label, as definedherein. In one method the mRNA is detected after immobilizing thenucleic acid to be examined on a solid support such as nylon membranesand hybridizing the probe with the sample. Following washing to removethe non-specifically bound probe, the label is detected. In anothermethod detection of the mRNA is performed in situ. In this methodpermeabilized cells or tissue samples are contacted with a detectablylabeled nucleic acid probe for sufficient time to allow the probe tohybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding a CA protein is detected by binding the digoxygenin withan anti-digoxygenin secondary antibody and developed with nitro bluetetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.

In a preferred embodiment, any of the three classes of proteins asdescribed herein (secreted, transmembrane or intracellular proteins) areused in diagnostic assays. The CA proteins, antibodies, nucleic acids,modified proteins and cells containing CA sequences are used indiagnostic assays. This can be done on an individual gene orcorresponding polypeptide level, or as sets of assays.

As described and defined herein, CA proteins find use as markers ofcancers, including lymphomas such as, but not limited to, Hodgkin's andnon-Hodgkin's lymphoma. Detection of these proteins in putative cancertissue or patients allows for a determination or diagnosis of the typeof cancer. Numerous methods known to those of ordinary skill in the artfind use in detecting cancers. In one embodiment, antibodies are used todetect CA proteins. A preferred method separates proteins from a sampleor patient by electrophoresis on a gel (typically a denaturing andreducing protein gel, but may be any other type of gel includingisoelectric focusing gels and the like). Following separation ofproteins, the CA protein is detected by immunoblotting with antibodiesraised against the CA protein. Methods of immunoblotting are well knownto those of ordinary skill in the art.

In another preferred method, antibodies to the CA protein find use in insitu imaging techniques. In this method cells are contacted with fromone to many antibodies to the CA protein(s). Following washing to removenon-specific antibody binding, the presence of the antibody orantibodies is detected. In one embodiment the antibody is detected byincubating with a secondary antibody that contains a detectable label.In another method the primary antibody to the CA protein(s) contains adetectable label. In another preferred embodiment each one of multipleprimary antibodies contains a distinct and detectable label. This methodfinds particular use in simultaneous screening for a plurality of CAproteins. As will be appreciated by one of ordinary skill in the art,numerous other histological imaging techniques are useful in theinvention.

In a preferred embodiment the label is detected in a fluorometer thathas the ability to detect and distinguish emissions of differentwavelengths. In addition, a fluorescence activated cell sorter (FACS)can be used in the method.

In another preferred embodiment, antibodies find use in diagnosingcancers from blood samples. As previously described, certain CA proteinsare secreted/circulating molecules. Blood samples, therefore, are usefulas samples to be probed or tested for the presence of secreted CAproteins. Antibodies can be used to detect the CA proteins by any of thepreviously described immunoassay techniques including ELISA,immunoblotting (Western blotting), immunoprecipitation, BIACOREtechnology and the like, as will be appreciated by one of ordinary skillin the art.

In a preferred embodiment, in situ hybridization of labeled CA nucleicacid probes to tissue arrays is done. For example, arrays of tissuesamples, including CA tissue and/or normal tissue, are made. In situhybridization as is known in the art can then be done.

It is understood that when comparing the expression fingerprints betweenan individual and a standard, the skilled artisan can make a diagnosisas well as a prognosis. It is further understood that the genes thatindicate diagnosis may differ from those that indicate prognosis.

In a preferred embodiment, the CA proteins, antibodies, nucleic acids,modified proteins and cells containing CA sequences are used inprognosis assays. As above, gene expression profiles can be generatedthat correlate to cancer, especially lymphoma, severity, in terms oflong term prognosis. Again, this may be done on either a protein or genelevel, with the use of genes being preferred. As above, the CA probesare attached to biochips for the detection and quantification of CAsequences in a tissue or patient. The assays proceed as outlined fordiagnosis.

Screening for CA-Targeted Drugs

In one embodiment, any of the CA sequences as described herein are usedin drug screening assays. The CA proteins, antibodies, nucleic acids,modified proteins and cells containing CA sequences are used in drugscreening assays or by evaluating the effect of drug candidates on a“gene expression profile” or expression profile of polypeptides. In oneembodiment, the expression profiles are used, preferably in conjunctionwith high throughput screening techniques to allow monitoring forexpression profile genes after treatment with a candidate agent,Zlokarnik, et al., Science 279, 84-8 (1998), Heid, et al., Genome Res.,6:986-994 (1996).

In another embodiment, the CA proteins, antibodies, nucleic acids,modified proteins and cells containing the native or modified CAproteins are used in screening assays. That is, the present inventionprovides novel methods for screening for compositions that modulate thecancer phenotype. As above, this can be done by screening for modulatorsof gene expression or for modulators of protein activity. Similarly,this may be done on an individual gene or protein level or by evaluatingthe effect of drug candidates on a “gene expression profile”. In apreferred embodiment, the expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring for expression profile genes after treatment with a candidateagent, see Zlokarnik, supra.

Having identified the CA genes herein, a variety of assays to evaluatethe effects of agents on gene expression may be executed. In a preferredembodiment, assays may be run on an individual gene or protein level.That is, having identified a particular gene as aberrantly regulated incancer, candidate bioactive agents may be screened to modulate thegene's regulation. “Modulation” thus includes both an increase and adecrease in gene expression or activity. The preferred amount ofmodulation will depend on the original change of the gene expression innormal versus tumor tissue, with changes of at least 10%, preferably50%, more preferably 100-300%, and in some embodiments 300-1000% orgreater. Thus, if a gene exhibits a 4 fold increase in tumor compared tonormal tissue, a decrease of about four fold is desired; a 10 folddecrease in tumor compared to normal tissue gives a 10 fold increase inexpression for a candidate agent is desired, etc. Alternatively, wherethe CA sequence has been altered but shows the same expression profileor an altered expression profile, the protein will be detected asoutlined herein.

As will be appreciated by those in the art, this may be done byevaluation at either the gene or the protein level; that is, the amountof gene expression may be monitored using nucleic acid probes and thequantification of gene expression levels, or, alternatively, the levelof the gene product itself can be monitored, for example through the useof antibodies to the CA protein and standard immunoassays.Alternatively, binding and bioactivity assays with the protein may bedone as outlined below.

In a preferred embodiment, gene expression monitoring is done and anumber of genes, i.e. an expression profile, is monitoredsimultaneously, although multiple protein expression monitoring can bedone as well.

In this embodiment, the CA nucleic acid probes are attached to biochipsas outlined herein for the detection and quantification of CA sequencesin a particular cell. The assays are further described below.

Generally, in a preferred embodiment, a candidate bioactive agent isadded to the cells prior to analysis. Moreover, screens are provided toidentify a candidate bioactive agent that modulates a particular type ofcancer, modulates CA proteins, binds to a CA protein, or interferesbetween the binding of a CA protein and an antibody.

The term “candidate bioactive agent” or “drug candidate” or grammaticalequivalents as used herein describes any molecule, e.g., protein,oligopeptide, small organic or inorganic molecule, polysaccharide,polynucleotide, etc., to be tested for bioactive agents that are capableof directly or indirectly altering either the cancer phenotype, bindingto and/or modulating the bioactivity of a CA protein, or the expressionof a CA sequence, including both nucleic acid sequences and proteinsequences. In a particularly preferred embodiment, the candidate agentsuppresses a CA phenotype, for example to a normal tissue fingerprint.Similarly, the candidate agent preferably suppresses a severe CAphenotype. Generally a plurality of assay mixtures are run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

In one aspect, a candidate agent will neutralize the effect of a CAprotein. By “neutralize” is meant that activity of a protein is eitherinhibited or counter acted against so as to have substantially no effecton a cell.

Candidate agents encompass numerous chemical classes, though typicallythey are organic or inorganic molecules, preferably small organiccompounds having a molecular weight of more than 100 and less than about2,500 Daltons. Preferred small molecules are less than 2000, or lessthan 1500 or less than 1000 or less than 500 D. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, or amidification to producestructural analogs.

In one embodiment, the candidate bioactive agents are proteins. By“protein” herein is meant at least two covalently attached amino acids,which includes proteins, polypeptides, oligopeptides and peptides. Theprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein means both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnorleucine are considered amino acids for the purposes of the invention.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The side chains may be in either the (R) or the (S)configuration. In the preferred embodiment, the amino acids are in the(S) or L-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of prokaryotic and eukaryotic proteins may be madefor screening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In another preferred embodiment, the candidate bioactive agents arepeptides of from about 5 to about 30 amino acids, with from about 5 toabout 20 amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation of nucleicacid binding domains, the creation of cysteines, for cross-linking,prolines for SH-3 domains, serines, threonines, tyrosines or histidinesfor phosphorylation sites, etc., or to purines, etc.

In one embodiment, the candidate bioactive agents are nucleic acids. Asdescribed generally for proteins, nucleic acid candidate bioactiveagents may be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. In another embodiment, the candidatebioactive agents are organic chemical moieties, a wide variety of whichare available in the literature.

In assays for testing alteration of the expression profile of one ormore CA genes, after the candidate agent has been added and the cellsallowed to incubate for some period of time, a nucleic acid samplecontaining the target sequences to be analyzed is prepared. The targetsequence is prepared using known techniques (e.g., converted from RNA tolabeled cDNA, as described above) and added to a suitable microarray.For example, an in vitro reverse transcription with labels covalentlyattached to the nucleosides is performed. Generally, the nucleic acidsare labeled with a label as defined herein, especially with biotin-FITCor PE, Cy3 and Cy5.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670,5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118,5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporatedby reference. In this embodiment, in general, the target nucleic acid isprepared as outlined above, and then added to the biochip comprising aplurality of nucleic acid probes, under conditions that allow theformation of a hybridization complex.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions asoutlined above. The assays are generally run under stringency conditionsthat allow formation of the label probe hybridization complex only inthe presence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration, pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents in the assays. These includereagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target. In addition, either solid phase or solution based(i.e., kinetic PCR) assays may be used.

Once the assay is run, the data are analyzed to determine the expressionlevels, and changes in expression levels as between states, ofindividual genes, forming a gene expression profile.

In a preferred embodiment, as for the diagnosis and prognosisapplications, having identified the differentially expressed gene(s) ormutated gene(s) important in any one state, screens can be run to testfor alteration of the expression of the CA genes individually. That is,screening for modulation of regulation of expression of a single genecan be done. Thus, for example, in the case of target genes whosepresence or absence is unique between two states, screening is done formodulators of the target gene expression.

In addition, screens can be done for novel genes that are induced inresponse to a candidate agent. After identifying a candidate agent basedupon its ability to suppress a CA expression pattern leading to a normalexpression pattern, or modulate a single CA gene expression profile soas to mimic the expression of the gene from normal tissue, a screen asdescribed above can be performed to identify genes that are specificallymodulated in response to the agent. Comparing expression profilesbetween normal tissue and agent treated CA tissue reveals genes that arenot expressed in normal tissue or CA tissue, but are expressed in agenttreated tissue. These agent specific sequences can be identified andused by any of the methods described herein for CA genes or proteins. Inparticular these sequences and the proteins they encode find use inmarking or identifying agent-treated cells. In addition, antibodies canbe raised against the agent-induced proteins and used to target noveltherapeutics to the treated CA tissue sample.

Thus, in one embodiment, a candidate agent is administered to apopulation of CA cells, that thus has an associated CA expressionprofile. By “administration” or “contacting” herein is meant that thecandidate agent is added to the cells in such a manner as to allow theagent to act upon the cell, whether by uptake and intracellular action,or by action at the cell surface. In some embodiments, nucleic acidencoding a proteinaceous candidate agent (i.e. a peptide) may be putinto a viral construct such as a retroviral construct and added to thecell, such that expression of the peptide agent is accomplished; see PCTUS97/01019, hereby expressly incorporated by reference.

Once the candidate agent has been administered to the cells, the cellscan be washed if desired and are allowed to incubate under preferablyphysiological conditions for some period of time. The cells are thenharvested and a new gene expression profile is generated, as outlinedherein.

Thus, for example, CA tissue may be screened for agents that reduce orsuppress the CA phenotype. A change in at least one gene of theexpression profile indicates that the agent has an effect on CAactivity. By defining such a signature for the CA phenotype, screens fornew drugs that alter the phenotype can be devised. With this approach,the drug target need not be known and need not be represented in theoriginal expression screening platform, nor does the level of transcriptfor the target protein need to change.

In a preferred embodiment, as outlined above, screens may be done onindividual genes and gene products (proteins). That is, havingidentified a particular differentially expressed gene as important in aparticular state, screening of modulators of either the expression ofthe gene or the gene product itself can be done. The gene products ofdifferentially expressed genes are sometimes referred to herein as “CAproteins” or “CAP”. The CAP may be a fragment, or alternatively, be thefull-length protein to the fragment encoded by the nucleic acids ofTables 1-16 (human genomic sequences of SEQ ID NOS: 4, 10, 18, 26, 34,44, 50, 60, 82, 88, 112, 122, 128, 136, 154, and 170, and sequences ofSEQ ID NOS: 5, 11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63,65, 67, 69, 71, 73, 75, 83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,113, 115, 117, 123, 129, 137, 139, 141, 143, 145, 147, 149, 155, 157,159, 161, 163, 165, 171, and 173 corresponding to the human mRNAsgenerated therefrom). In a preferred embodiment, the CAP is selectedfrom the human protein sequences shown in Tables 1-16 (of SEQ ID NOS: 6,12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70, 72,74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116, 118,124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162, 164,166, 172, and 174). In another embodiment, the sequences are sequencevariants as further described herein.

Preferably, the CAP is a fragment approximately 14 to 24 amino acids inlength. More preferably the fragment is a soluble fragment. Preferably,the fragment includes a non-transmembrane region. In a preferredembodiment, the fragment has an N-terminal Cys to aid in solubility. Inone embodiment, the C-terminus of the fragment is kept as a free acidand the N-terminus is a free amine to aid in coupling, e.g., to acysteine.

In one embodiment the CA proteins are conjugated to an immunogenic agentas discussed herein. In one embodiment the CA protein is conjugated toBSA.

In a preferred embodiment, screening is done to alter the biologicalfunction of the expression product of the CA gene. Again, havingidentified the importance of a gene in a particular state, screening foragents that bind and/or modulate the biological activity of the geneproduct can be run as is more fully outlined below.

In a preferred embodiment, screens are designed to first find candidateagents that can bind to CA proteins, and then these agents may be usedin assays that evaluate the ability of the candidate agent to modulatethe CAP activity and the cancer phenotype. Thus, as will be appreciatedby those in the art, there are a number of different assays that may berun; binding assays and activity assays.

In a preferred embodiment, binding assays are done. In general, purifiedor isolated gene product is used; that is, the gene products of one ormore CA nucleic acids are made. In general, this is done as is known inthe art. For example, antibodies are generated to the protein geneproducts, and standard immunoassays are run to determine the amount ofprotein present. Alternatively, cells comprising the CA proteins can beused in the assays.

Thus, in a preferred embodiment, the methods comprise combining a CAprotein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the CA protein. Preferred embodiments utilize thehuman or mouse CA protein, although other mammalian proteins may also beused, for example for the development of animal models of human disease.In some embodiments, as outlined herein, variant or derivative CAproteins may be used.

Generally, in a preferred embodiment of the methods herein, the CAprotein or the candidate agent is non-diffusably bound to an insolublesupport having isolated sample receiving areas (e.g. a microtiter plate,an array, etc.). The insoluble support may be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes and beads. These are typically madeof glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, Teflon®, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples.

The particular manner of binding of the composition is not crucial solong as it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. Following bindingof the protein or agent, excess unbound material is removed by washing.The sample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

In a preferred embodiment, the CA protein is bound to the support, and acandidate bioactive agent is added to the assay. Alternatively, thecandidate agent is bound to the support and the CA protein is added.Novel binding agents include specific antibodies, non-natural bindingagents identified in screens of chemical libraries, peptide analogs,etc. Of particular interest are screening assays for agents that have alow toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to theCA protein may be done in a number of ways. In a preferred embodiment,the candidate bioactive agent is labeled, and binding determineddirectly. For example, this may be done by attaching all or a portion ofthe CA protein to a solid support, adding a labeled candidate agent (forexample a fluorescent label), washing off excess reagent, anddetermining whether the label is present on the solid support. Variousblocking and washing steps may be utilized as is known in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophore for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. CA protein), such as an antibody, peptide, bindingpartner, ligand, etc. Under certain circumstances, there may becompetitive binding as between the bioactive agent and the bindingmoiety, with the binding moiety displacing the bioactive agent.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high throughput screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the CAprotein and thus is capable of binding to, and potentially modulating,the activity of the CA protein. In this embodiment, either component canbe labeled. Thus, for example, if the competitor is labeled, thepresence of label in the wash solution indicates displacement by theagent. Alternatively, if the candidate bioactive agent is labeled, thepresence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the CA protein with a higher affinity. Thus, if thecandidate bioactive agent is labeled, the presence of the label on thesupport, coupled with a lack of competitor binding, may indicate thatthe candidate agent is capable of binding to the CA protein.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating the activityof the CA proteins. In this embodiment, the methods comprise combining aCA protein and a competitor in a first sample. A second sample comprisesa candidate bioactive agent, a CA protein and a competitor. The bindingof the competitor is determined for both samples, and a change, ordifference in binding between the two samples indicates the presence ofan agent capable of binding to the CA protein and potentially modulatingits activity. That is, if the binding of the competitor is different inthe second sample relative to the first sample, the agent is capable ofbinding to the CA protein.

Alternatively, a preferred embodiment utilizes differential screening toidentify drug candidates that bind to the native CA protein, but cannotbind to modified CA proteins. The structure of the CA protein may bemodeled, and used in rational drug design to synthesize agents thatinteract with that site. Drug candidates that affect CA bioactivity arealso identified by screening drugs for the ability to either enhance orreduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Screening for agents that modulate the activity of CA proteins may alsobe done. In a preferred embodiment, methods for screening for abioactive agent capable of modulating the activity of CA proteinscomprise the steps of adding a candidate bioactive agent to a sample ofCA proteins, as above, and determining an alteration in the biologicalactivity of CA proteins. “Modulating the activity of a CA protein”includes an increase in activity, a decrease in activity, or a change inthe type or kind of activity present. Thus, in this embodiment, thecandidate agent should both bind to CA proteins (although this may notbe necessary), and alter its biological or biochemical activity asdefined herein. The methods include both in vitro screening methods, asare generally outlined above, and in vivo screening of cells foralterations in the presence, distribution, activity or amount of CAproteins.

Thus, in this embodiment, the methods comprise combining a CA sample anda candidate bioactive agent, and evaluating the effect on CA activity.By “CA activity” or grammatical equivalents herein is meant one of theCA protein's biological activities, including, but not limited to, itsrole in tumorigenesis, including cell division, preferably in lymphatictissue, cell proliferation, tumor growth and transformation of cells. Inone embodiment, CA activity includes activation of or by a proteinencoded by a nucleic acid of Tables 1-16. An inhibitor of CA activity isthe inhibition of any one or more CA activities.

In a preferred embodiment, the activity of the CA protein is increased;in another preferred embodiment, the activity of the CA protein isdecreased. Thus, bioactive agents that are antagonists are preferred insome embodiments, and bioactive agents that are agonists may bepreferred in other embodiments.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of a CA protein.The methods comprise adding a candidate bioactive agent, as definedabove, to a cell comprising CA proteins. Preferred cell types includealmost any cell. The cells contain a recombinant nucleic acid thatencodes a CA protein. In a preferred embodiment, a library of candidateagents is tested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, for examplehormones, antibodies, peptides, antigens, cytokines, growth factors,action potentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e. cell-cell contacts). Inanother example, the determinations are determined at different stagesof the cell cycle process.

In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the CA protein.

APPLICATIONS OF THE INVENTION

In one embodiment, a method of inhibiting cancer cell division isprovided. In another embodiment, a method of inhibiting tumor growth isprovided. In a further embodiment, methods of treating cells orindividuals with cancer are provided.

The method comprises administration of a cancer inhibitor. In particularembodiments, the cancer inhibitor is an antisense molecule, apharmaceutical composition, a therapeutic agent or small molecule, or amonoclonal, polyclonal, chimeric or humanized antibody. In particularembodiments, a therapeutic agent is coupled with a an antibody,preferable a monoclonal antibody.

In other embodiments, methods for detection or diagnosis of cancer cellsin an individual are provided. In particular embodiments, thediagnostic/detection agent is a small molecule that preferentially bindsto a CAP according to the invention. In one embodiment, thediagnostic/detection agent is an antibody, preferably a monoclonalantibody, preferably linked to a detectable agent.

In other embodiments of the invention, animal models and transgenicanimals are provided, which find use in generating animal models ofcancers, particularly lymphomas and carcinomas.

(a) Antisense Molecules

In one embodiment, the cancer inhibitor is an antisense molecule.Antisense molecules as used herein include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 14 nucleotides, preferably from about14 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988)and van der Krol et al., BioTechniques 6:958, (1988).

Antisense molecules can be modified or unmodified RNA, DNA, or mixedpolymer oligonucleotides. These molecules function by specificallybinding to matching sequences resulting in inhibition of peptidesynthesis (Wu-Pong, November 1994, BioPharm, 20-33) either by stericblocking or by activating an RNase H enzyme. Antisense molecules canalso alter protein synthesis by interfering with RNA processing ortransport from the nucleus into the cytoplasm (Mukhopadhyay & Roth,1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding ofsingle stranded DNA to RNA can result in nuclease-mediated degradationof the heteroduplex (Wu-Pong, supra). Backbone modified DNA chemistrywhich have thus far been shown to act as substrates for RNase H arephosphorothioates, phosphorodithioates, borontrifluoridates, and2′-arabino and 2′-fluoro arabino-containing oligonucleotides.

Antisense molecules may be introduced into a cell containing the targetnucleotide sequence by formation of a conjugate with a ligand bindingmolecule, as described in WO 91/04753. Suitable ligand binding moleculesinclude, but are not limited to, cell surface receptors, growth factors,other cytokines, or other ligands that bind to cell surface receptors.Preferably, conjugation of the ligand binding molecule does notsubstantially interfere with the ability of the ligand binding moleculeto bind to its corresponding molecule or receptor, or block entry of thesense or antisense oligonucleotide or its conjugated version into thecell. Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. It is understood that the use of antisense molecules or knockout and knock in models may also be used in screening assays asdiscussed above, in addition to methods of treatment.

(b) RNA Interference

RNA interference refers to the process of sequence-specific posttranscriptional gene silencing in animals mediated by short interferingRNAs (siRNA) (Fire et al., Nature, 391, 806 (1998)). The correspondingprocess in plants is referred to as post transcriptional gene silencingor RNA silencing and is also referred to as quelling in fungi. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L. (reviewedin Sharp, P.A., RNA interference-2001, Genes & Development 15:485-490(2001)).

Small interfering RNAs (siRNAs) are powerful sequence-specific reagentsdesigned to suppress the expression of genes in cultured mammalian cellsthrough a process known as RNA interference (RNAi). Elbashir, S. M. etal. Nature 411:494-498 (2001); Caplen, N. J. et al. Proc. Natl. Acad.Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J. Cell Sci.114:4557-4565 (2001). The term “short interfering RNA” or “siRNA” refersto a double stranded nucleic acid molecule capable of RNA interference“RNAi”, (see Kreutzer et al., WO 00/44895; Zernicka-Goetz et al. WO01/36646; Fire, WO 99/32619; Mello and Fire, WO 01/29058). As usedherein, siRNA molecules are limited to RNA molecules but furtherencompasses chemically modified nucleotides and non-nucleotides. siRNAgene-targeting experiments have been carried out by transient siRNAtransfer into cells (achieved by such classic methods asliposome-mediated transfection, electroporation, or microinjection).

Molecules of siRNA are 21- to 23-nucleotide RNAs, with characteristic 2-to 3-nucleotide 3′-overhanging ends resembling the RNase III processingproducts of long double-stranded RNAs (dsRNAs) that normally initiateRNAi. When introduced into a cell, they assemble withyet-to-be-identified proteins of an endonuclease complex (RNA-inducedsilencing complex), which then guides target mRNA cleavage. As aconsequence of degradation of the targeted mRNA, cells with a specificphenotype characteristic of suppression of the corresponding proteinproduct are obtained. The small size of siRNAs, compared withtraditional antisense molecules, prevents activation of thedsRNA-inducible interferon system present in mammalian cells. Thisavoids the nonspecific phenotypes normally produced by dsRNA larger than30 base pairs in somatic cells.

Intracellular transcription of small RNA molecules is achieved bycloning the siRNA templates into RNA polymerase III (Pol III)transcription units, which normally encode the small nuclear RNA (snRNA)U6 or the human RNase P RNA H1. Two approaches have been developed forexpressing siRNAs: in the first, sense and antisense strandsconstituting the siRNA duplex are transcribed by individual promoters(Lee, N. S. et al. Nat. Biotechnol. 20, 500-505 (2002). Miyagishi, M. &Taira, K. Nat. Biotechnol. 20, 497-500 (2002).); in the second, siRNAsare expressed as fold-back stem-loop structures that give rise to siRNAsafter intracellular processing (Paul, C. P. et al. Nat. Biotechnol.20:505-508 (2002)). The endogenous expression of siRNAs from introducedDNA templates is thought to overcome some limitations of exogenous siRNAdelivery, in particular the transient loss of phenotype. U6 and H1 RNApromoters are members of the type III class of Pol III promoters.(Paule, M. R. & White, R. J. Nucleic Acids Res. 28, 1283-1298 (2000)).

Co-expression of sense and antisense siRNAs mediate silencing of targetgenes, whereas expression of sense or antisense siRNA alone do notgreatly affect target gene expression. Transfection of plasmid DNA,rather than synthetic siRNAs, may appear advantageous, considering thedanger of RNase contamination and the costs of chemically synthesizedsiRNAs or siRNA transcription kits. Stable expression of siRNAs allowsnew gene therapy applications, such as treatment of persistent viralinfections. Considering the high specificity of siRNAs, the approachalso allows the targeting of disease-derived transcripts with pointmutations, such as RAS or TP53 oncogene transcripts, without alterationof the remaining wild-type allele. Finally, by high-throughput sequenceanalysis of the various genomes, the DNA-based methodology may also be acost-effective alternative for automated genome-wide loss-of-functionphenotypic analysis, especially when combined with miniaturizedarray-based phenotypic screens. (Ziauddin, J. & Sabatini, D. M. Nature411:107-110 (2001)).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409:363(2001)). Short interfering RNAs derived from dicer activity aretypically about 21-23 nucleotides in length and comprise about 19 basepair duplexes. Dicer has also been implicated in the excision of 21 and22 nucleotide small temporal RNAs (stRNA) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., Science, 293, 834 (2001)). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., Genes Dev.,15, 188 (2001)).

This invention provides an expression system comprising an isolatednucleic acid molecule comprising a sequence capable of specificallyhybridizing to the CA sequences. In an embodiment, the nucleic acidmolecule is capable of inhibiting the expression of the CA protein. Amethod of inhibiting expression of CA inside a cell by a vector-directedexpression of a short RNA which short RNA can fold in itself and createa double strand RNA having CA mRNA sequence identity and able to triggerposttranscriptional gene silencing, or RNA interference (RNAi), of theCA gene inside the cell. In another method a short double strand RNAhaving CA mRNA sequence identity is delivered inside the cell to triggerposttranscriptional gene silencing, or RNAi, of the CA gene. In variousembodiments, the nucleic acid molecule is at least a 7 mer, at least a10 mer, or at least a 20 mer. In a further embodiment, the sequence isunique.

(c) Pharmaceutical Compositions

Pharmaceutical compositions encompassed by the present invention includeas active agent, the polypeptides, polynucleotides, antisenseoligonucleotides, or antibodies of the invention disclosed herein in atherapeutically effective amount. An “effective amount” is an amountsufficient to effect beneficial or desired results, including clinicalresults. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofan adenoviral vector is an amount that is sufficient to palliate,ameliorate, stabilize, reverse, slow or delay the progression of thedisease state.

The compositions can be used to treat cancer as well as metastases ofprimary cancer. In addition, the pharmaceutical compositions can be usedin conjunction with conventional methods of cancer treatment, e.g., tosensitize tumors to radiation or conventional chemotherapy. The terms“treatment”, “treating”, “treat” and the like are used herein togenerally refer to obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete stabilization or cure fora disease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal,particularly a human, and includes: (a) preventing the disease orsymptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

Where the pharmaceutical composition comprises an antibody thatspecifically binds to a gene product encoded by a differentiallyexpressed polynucleotide, the antibody can be coupled to a drug fordelivery to a treatment site or coupled to a detectable label tofacilitate imaging of a site comprising cancer cells, such as prostatecancer cells. Methods for coupling antibodies to drugs and detectablelabels are well known in the art, as are methods for imaging usingdetectable labels.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent to treat, ameliorate, or prevent a desireddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The effect can be detected by, for example,chemical markers or antigen levels. Therapeutic effects also includereduction in physical symptoms, such as decreased body temperature. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and extent of the condition, and thetherapeutics or combination of therapeutics selected for administration.The effective amount for a given situation is determined by routineexperimentation and is within the judgment of the clinician. Forpurposes of the present invention, an effective dose will generally befrom about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 50mg/kg or about 0.05 mg/kg to about 10 mg/kg of the compositions of thepresent invention in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which can be administered without undue toxicity.Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers in therapeuticcompositions can include liquids such as water, saline, glycerol andethanol. Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, can also be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable salts can also be present in the pharmaceutical composition,e.g., mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients is available inRemington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro,Lippincott, Williams, & Wilkins.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

The pharmaceutical compositions of the present invention comprise a CAprotein in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts.“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host, aspreviously described. The agents may be administered in a variety ofways, orally, parenterally e.g., subcutaneously, intraperitoneally,intravascularly, etc. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100% wgt/vol. Once formulated, the compositions contemplated by theinvention can be (1) administered directly to the subject (e.g., aspolynucleotide, polypeptides, small molecule agonists or antagonists,and the like); or (2) delivered ex vivo, to cells derived from thesubject (e.g., as in ex vivo gene therapy). Direct delivery of thecompositions will generally be accomplished by parenteral injection,e.g., subcutaneously, intraperitoneally, intravenously orintramuscularly, intratumoral or to the interstitial space of a tissue.Other modes of administration include oral and pulmonary administration,suppositories, and transdermal applications, needles, and gene guns orhyposprays. Dosage treatment can be a single dose schedule or a multipledose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cellsinto a subject are known in the art and described in e.g., InternationalPublication No. WO 93/14778. Examples of cells useful in ex vivoapplications include, for example, stem cells, particularlyhematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.Generally, delivery of nucleic acids for both ex vivo and in vitroapplications can be accomplished by, for example, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei, all well known in the art.

Once differential expression of a gene corresponding to a CApolynucleotide described herein has been found to correlate with aproliferative disorder, such as neoplasia, dysplasia, and hyperplasia,the disorder can be amenable to treatment by administration of atherapeutic agent based on the provided polynucleotide, correspondingpolypeptide or other corresponding molecule (e.g., antisense, ribozyme,etc.). In other embodiments, the disorder can be amenable to treatmentby administration of a small molecule drug that, for example, serves asan inhibitor (antagonist) of the function of the encoded gene product ofa gene having increased expression in cancerous cells relative to normalcells or as an agonist for gene products that are decreased inexpression in cancerous cells (e.g., to promote the activity of geneproducts that act as tumor suppressors).

The dose and the means of administration of the inventive pharmaceuticalcompositions are determined based on the specific qualities of thetherapeutic composition, the condition, age, and weight of the patient,the progression of the disease, and other relevant factors. For example,administration of polynucleotide therapeutic compositions agentsincludes local or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. Preferably, the therapeutic polynucleotide compositioncontains an expression construct comprising a promoter operably linkedto a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nt ofthe polynucleotide disclosed herein. Various methods can be used toadminister the therapeutic composition directly to a specific site inthe body. For example, a small metastatic lesion is located and thetherapeutic composition injected several times in several differentlocations within the body of tumor. Alternatively, arteries that serve atumor are identified, and the therapeutic composition injected into suchan artery, in order to deliver the composition directly into the tumor.A tumor that has a necrotic center is aspirated and the compositioninjected directly into the now empty center of the tumor. An antisensecomposition is directly administered to the surface of the tumor, forexample, by topical application of the composition. X-ray imaging isused to assist in certain of the above delivery methods.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, subgenomic polynucleotides, or antibodies to specifictissues can also be used. Receptor-mediated DNA delivery techniques aredescribed in, for example, Findeis et al., Trends Biotechnol. (1993)11:202; Chiou et al., Gene Therapeutics: Methods And Applications OfDirect Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol.Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke etal., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol.Chem. (1991) 266:338. Therapeutic compositions containing apolynucleotide are administered in a range of about 100 ng to about 200mg of DNA for local administration in a gene therapy protocol.Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAcan also be used during a gene therapy protocol. Factors such as methodof action (e.g., for enhancing or inhibiting levels of the encoded geneproduct) and efficacy of transformation and expression areconsiderations that will affect the dosage required for ultimateefficacy of the antisense subgenomic polynucleotides. Where greaterexpression is desired over a larger area of tissue, larger amounts ofantisense subgenomic polynucleotides or the same amounts re-administeredin a successive protocol of administrations, or several administrationsto different adjacent or close tissue portions of, for example, a tumorsite, may be required to effect a positive therapeutic outcome. In allcases, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect.

The therapeutic polynucleotides and polypeptides of the presentinvention can be delivered using gene delivery vehicles. The genedelivery vehicle can be of viral or non-viral origin (see generally,Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy(1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt,Nature Genetics (1994) 6:148). Expression of such coding sequences canbe induced using endogenous mammalian or heterologous promoters.Expression of the coding sequence can be either constitutive orregulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat.No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805),alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forestvirus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCCVR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCCVR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV)vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938;WO 95/11984 and WO 95/00655). Administration of DNA linked to killedadenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can alsobe employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO97/42338) and nucleic charge neutralization or fusion with cellmembranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in WO 90/11092 and U.S. Pat. No.5,580,859. Liposomes that can act as gene delivery vehicles aredescribed in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO91/14445; and EP 0524968. Additional approaches are described in Philip,Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad.Sci. (1994) 91:1581.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad. Sci. USA (1994) 91(24):11581. Moreover, the coding sequence andthe product of expression of such can be delivered through deposition ofphotopolymerized hydrogel materials or use of ionizing radiation (see,e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation foractivating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO92/11033).

The administration of the CA proteins and modulators of the presentinvention can be done in a variety of ways as discussed above,including, but not limited to, orally, subcutaneously, intravenously,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. In someinstances, for example, in the treatment of wounds and inflammation, theCA proteins and modulators may be directly applied as a solution orspray.

In a preferred embodiment, CA proteins and modulators are administeredas therapeutic agents, and can be formulated as outlined above.Similarly, CA genes (including both the full-length sequence, partialsequences, or regulatory sequences of the CA coding regions) can beadministered in gene therapy applications, as is known in the art. TheseCA genes can include antisense applications, either as gene therapy(i.e. for incorporation into the genome) or as antisense compositions,as will be appreciated by those in the art.

Thus, in one embodiment, methods of modulating CA gene activity in cellsor organisms are provided. In one embodiment, the methods compriseadministering to a cell an anti-CA antibody that reduces or eliminatesthe biological activity of an endogenous CA protein. Alternatively, themethods comprise administering to a cell or organism a recombinantnucleic acid encoding a CA protein. As will be appreciated by those inthe art, this may be accomplished in any number of ways. In a preferredembodiment, for example when the CA sequence is down-regulated incancer, the activity of the CA gene product is increased by increasingthe amount of CA expression in the cell, for example by overexpressingthe endogenous CA gene or by administering a gene encoding the CAsequence, using known gene-therapy techniques. In a preferredembodiment, the gene therapy techniques include the incorporation of theexogenous gene using enhanced homologous recombination (EHR), forexample as described in PCT/US93/03868, hereby incorporated by referencein its entirety. Alternatively, for example when the CA sequence isup-regulated in cancer, the activity of the endogenous CA gene isdecreased, for example by the administration of a CA antisense nucleicacid.

(d) Vaccines

In a preferred embodiment, CA genes are administered as DNA vaccines,either single genes or combinations of CA genes. Naked DNA vaccines aregenerally known in the art. Brower, Nature Biotechnology, 16:1304-1305(1998).

In one embodiment, CA genes of the present invention are used as DNAvaccines. Methods for the use of genes as DNA vaccines are well known toone of ordinary skill in the art, and include placing a CA gene orportion of a CA gene under the control of a promoter for expression in apatient with cancer. The CA gene used for DNA vaccines can encodefull-length CA proteins, but more preferably encodes portions of the CAproteins including peptides derived from the CA protein. In a preferredembodiment a patient is immunized with a DNA vaccine comprising aplurality of nucleotide sequences derived from a CA gene. Similarly, itis possible to immunize a patient with a plurality of CA genes orportions thereof. Without being bound by theory, expression of thepolypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced that recognize and destroy oreliminate cells expressing CA proteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the CA polypeptideencoded by the DNA vaccine. Additional or alternative adjuvants areknown to those of ordinary skill in the art and find use in theinvention.

(e) Antibodies

In one embodiment, a cancer inhibitor is an antibody as discussed above.In one embodiment, the CA proteins of the present invention may be usedto generate polyclonal and monoclonal antibodies to CA proteins, whichare useful as described herein. Similarly, the CA proteins can becoupled, using standard technology, to affinity chromatography columns.These columns may then be used to purify CA antibodies. In a preferredembodiment, the antibodies are generated to epitopes unique to a CAprotein; that is, the antibodies show little or no cross-reactivity toother proteins. These antibodies find use in a number of applications.For example, the CA antibodies may be coupled to standard affinitychromatography columns and used to purify CA proteins. The antibodiesmay also be used therapeutically as blocking polypeptides, as outlinedabove, since they will specifically bind to the CA protein.

The present invention further provides methods for detecting thepresence of and/or measuring a level of a polypeptide in a biologicalsample, which CA polypeptide is encoded by a CA polynucleotide that isdifferentially expressed in a cancer cell, using an antibody specificfor the encoded polypeptide. The methods generally comprise: a)contacting the sample with an antibody specific for a polypeptideencoded by a CA polynucleotide that is differentially expressed in aprostate cancer cell; and b) detecting binding between the antibody andmolecules of the sample.

Detection of specific binding of the antibody specific for the encodedcancer-associated polypeptide, when compared to a suitable control is anindication that encoded polypeptide is present in the sample. Suitablecontrols include a sample known not to contain the encoded CApolypeptide or known not to contain elevated levels of the polypeptide;such as normal tissue, and a sample contacted with an antibody notspecific for the encoded polypeptide, e.g., an anti-idiotype antibody. Avariety of methods to detect specific antibody-antigen interactions areknown in the art and can be used in the method, including, but notlimited to, standard immunohistological methods, immunoprecipitation, anenzyme immunoassay, and a radioimmunoassay. In general, the specificantibody will be detectably labeled, either directly or indirectly.Direct labels include radioisotopes; enzymes whose products aredetectable (e.g., luciferase, 3-galactosidase, and the like);fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin, aequorin (green fluorescent protein), andthe like. The antibody may be attached (coupled) to an insolublesupport, such as a polystyrene plate or a bead. Indirect labels includesecond antibodies specific for antibodies specific for the encodedpolypeptide (“first specific antibody”), wherein the second antibody islabeled as described above; and members of specific binding pairs, e.g.,biotin-avidin, and the like. The biological sample may be brought intocontact with and immobilized on a solid support or carrier, such asnitrocellulose, that is capable of immobilizing cells, cell particles,or soluble proteins. The support may then be washed with suitablebuffers, followed by contacting with a detectably-labeled first specificantibody. Detection methods are known in the art and will be chosen asappropriate to the signal emitted by the detectable label. Detection isgenerally accomplished in comparison to suitable controls, and toappropriate standards.

In some embodiments, the methods are adapted for use in vivo, e.g., tolocate or identify sites where cancer cells are present. In theseembodiments, a detectably-labeled moiety, e.g., an antibody, which isspecific for a cancer-associated polypeptide is administered to anindividual (e.g., by injection), and labeled cells are located usingstandard imaging techniques, including, but not limited to, magneticresonance imaging, computed tomography scanning, and the like. In thismanner, cancer cells are differentially labeled.

(f) Detection and Diagnosis of Cancers

Without being bound by theory, it appears that the various CA sequencesare important in cancers. Accordingly, disorders based on mutant orvariant CA genes may be determined. In one embodiment, the inventionprovides methods for identifying cells containing variant CA genescomprising determining all or part of the sequence of at least oneendogenous CA genes in a cell. As will be appreciated by those in theart, this may be done using any number of sequencing techniques. In apreferred embodiment, the invention provides methods of identifying theCA genotype of an individual comprising determining all or part of thesequence of at least one CA gene of the individual. This is generallydone in at least one tissue of the individual, and may include theevaluation of a number of tissues or different samples of the sametissue. The method may include comparing the sequence of the sequencedCA gene to a known CA gene, i.e., a wild-type gene. As will beappreciated by those in the art, alterations in the sequence of some CAgenes can be an indication of either the presence of the disease, orpropensity to develop the disease, or prognosis evaluations.

The sequence of all or part of the CA gene can then be compared to thesequence of a known CA gene to determine if any differences exist. Thiscan be done using any number of known homology programs, such asBestfit, etc. In a preferred embodiment, the presence of a difference inthe sequence between the CA gene of the patient and the known CA gene isindicative of a disease state or a propensity for a disease state, asoutlined herein.

In a preferred embodiment, the CA genes are used as probes to determinethe number of copies of the CA gene in the genome. For example, somecancers exhibit chromosomal deletions or insertions, resulting in analteration in the copy number of a gene.

In another preferred embodiment CA genes are used as probes to determinethe chromosomal location of the CA genes. Information such aschromosomal location finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in CA gene loci.

The present invention provides methods of using the polynucleotidesdescribed herein for detecting cancer cells, facilitating diagnosis ofcancer and the severity of a cancer (e.g., tumor grade, tumor burden,and the like) in a subject, facilitating a determination of theprognosis of a subject, and assessing the responsiveness of the subjectto therapy (e.g., by providing a measure of therapeutic effect through,for example, assessing tumor burden during or following achemotherapeutic regimen). Detection can be based on detection of apolynucleotide that is differentially expressed in a cancer cell, and/ordetection of a polypeptide encoded by a polynucleotide that isdifferentially expressed in a cancer cell. The detection methods of theinvention can be conducted in vitro or in vivo, on isolated cells, or inwhole tissues or a bodily fluid e.g., blood, plasma, serum, urine, andthe like).

In some embodiments, methods are provided for detecting a cancer cell bydetecting expression in the cell of a transcript that is differentiallyexpressed in a cancer cell. Any of a variety of known methods can beused for detection, including, but not limited to, detection of atranscript by hybridization with a polynucleotide that hybridizes to apolynucleotide that is differentially expressed in a prostate cancercell; detection of a transcript by a polymerase chain reaction usingspecific oligonucleotide primers; in situ hybridization of a cell usingas a probe a polynucleotide that hybridizes to a gene that isdifferentially expressed in a prostate cancer cell. The methods can beused to detect and/or measure mRNA levels of a gene that isdifferentially expressed in a cancer cell. In some embodiments, themethods comprise: a) contacting a sample with a polynucleotide thatcorresponds to a differentially expressed gene described herein underconditions that allow hybridization; and b) detecting hybridization, ifany.

Detection of differential hybridization, when compared to a suitablecontrol, is an indication of the presence in the sample of apolynucleotide that is differentially expressed in a cancer cell.Appropriate controls include, for example, a sample that is known not tocontain a polynucleotide that is differentially expressed in a cancercell, and use of a labeled polynucleotide of the same “sense” as thepolynucleotide that is differentially expressed in the cancer cell.Conditions that allow hybridization are known in the art, and have beendescribed in more detail above. Detection can also be accomplished byany known method, including, but not limited to, in situ hybridization,PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR),TMA, bDNA, and Nasbau and “Northern” or RNA blotting, or combinations ofsuch techniques, using a suitably labeled polynucleotide. A variety oflabels and labeling methods for polynucleotides are known in the art andcan be used in the assay methods of the invention. Specificity ofhybridization can be determined by comparison to appropriate controls.

Polynucleotides generally comprising at least 10 nt, at least 12 nt orat least 15 contiguous nucleotides of a polynucleotide provided herein,such as, for example, those having the sequence as depicted in Tables1-16, are used for a variety of purposes, such as probes for detectionof and/or measurement of, transcription levels of a polynucleotide thatis differentially expressed in a prostate cancer cell. As will bereadily appreciated by the ordinarily skilled artisan, the probe can bedetectably labeled and contacted with, for example, an array comprisingimmobilized polynucleotides obtained from a test sample (e.g., mRNA).Alternatively, the probe can be immobilized on an array and the testsample detectably labeled. These and other variations of the methods ofthe invention are well within the skill in the art and are within thescope of the invention.

Nucleotide probes are used to detect expression of a gene correspondingto the provided polynucleotide. In Northern blots, mRNA is separatedelectrophoretically and contacted with a probe. A probe is detected ashybridizing to an mRNA species of a particular size. The amount ofhybridization can be quantitated to determine relative amounts ofexpression, for example under a particular condition. Probes are usedfor in situ hybridization to cells to detect expression. Probes can alsobe used in vivo for diagnostic detection of hybridizing sequences.Probes are typically labeled with a radioactive isotope. Other types ofdetectable labels can be used such as chromophores, fluorophores, andenzymes. Other examples of nucleotide hybridization assays are describedin WO92/02526 and U.S. Pat. No. 5,124,246.

PCR is another means for detecting small amounts of target nucleic acids(see, e.g., Mullis et al., Meth. Enzymol. (1987) 155:335; U.S. Pat. No.4,683,195; and U.S. Pat. No. 4,683,202). Two primer oligonucleotidesthat hybridize with the target nucleic acids are used to prime thereaction. The primers can be composed of sequence within or 3′ and 5′ tothe CA polynucleotides disclosed herein. Alternatively, if the primersare 3′ and 5′ to these polynucleotides, they need not hybridize to themor the complements. After amplification of the target with athermostable polymerase, the amplified target nucleic acids can bedetected by methods known in the art, e.g., Southern blot. mRNA or cDNAcan also be detected by traditional blotting techniques (e.g., Southernblot, Northern blot, etc.) described in Sambrook et al., “MolecularCloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory,1989) (e.g., without PCR amplification). In general, mRNA or cDNAgenerated from mRNA using a polymerase enzyme can be purified andseparated using gel electrophoresis, and transferred to a solid support,such as nitrocellulose. The solid support is exposed to a labeled probe,washed to remove any unhybridized probe, and duplexes containing thelabeled probe are detected.

Methods using PCR amplification can be performed on the DNA from asingle cell, although it is convenient to use at least about 10⁵ cells.The use of the polymerase chain reaction is described in Saiki et al.(1985) Science 239:487, and a review of current techniques may be foundin Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press1989, pp. 14.2-14.33. A detectable label may be included in theamplification reaction. Suitable detectable labels include fluorochromes(e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red,phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein,6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)),radioactive labels, (e.g. ³²P, ³⁵S, ³H, etc.), and the like. The labelmay be a two stage system, where the polynucleotides is conjugated tobiotin, haptens, etc. having a high affinity binding partner, e.g.avidin, specific antibodies, etc., where the binding partner isconjugated to a detectable label. The label may be conjugated to one orboth of the primers. Alternatively, the pool of nucleotides used in theamplification is labeled, so as to incorporate the label into theamplification product.

The detection methods can be provided as part of a kit. Thus, theinvention further provides kits for detecting the presence and/or alevel of a polynucleotide that is differentially expressed in a cancercell (e.g., by detection of an mRNA encoded by the differentiallyexpressed gene of interest), and/or a polypeptide encoded thereby, in abiological sample. Procedures using these kits can be performed byclinical laboratories, experimental laboratories, medical practitioners,or private individuals. The kits of the invention for detecting apolypeptide encoded by a polynucleotide that is differentially expressedin a cancer cell may comprise a moiety that specifically binds thepolypeptide, which may be an antibody that binds the polypeptide orfragment thereof. The kits of the invention used for detecting apolynucleotide that is differentially expressed in a prostate cancercell may comprise a moiety that specifically hybridizes to such apolynucleotide. The kit may optionally provide additional componentsthat are useful in the procedure, including, but not limited to,buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, standards, instructions, and interpretiveinformation. Accordingly, the present invention provides kits fordetecting prostate cancer comprising at least one of polynucleotideshaving the sequence as shown in Tables 1-16 or fragments thereof.

The present invention further relates to methods of detecting/diagnosinga neoplastic or preneoplastic condition in a mammal (for example, ahuman). “Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and therametrics(e.g., monitoring a subject's condition to provide information as to theeffect or efficacy of therapy).

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

An “effective amount” is an amount sufficient to effect beneficial ordesired results, including clinical results. An effective amount can beadministered in one or more administrations.

A “cell sample” encompasses a variety of sample types obtained from anindividual and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “cell sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”,“tumor cells”, “cancer” and “cancer cells”, (used interchangeably) referto cells which exhibit relatively autonomous growth, so that theyexhibit an aberrant growth phenotype characterized by a significant lossof control of cell proliferation (i.e., de-regulated cell division).Neoplastic cells can be malignant or benign.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Othersubjects may include cattle, dogs, cats, guinea pigs, rabbits, rats,mice, horses, and so on. Examples of conditions that can bedetected/diagnosed in accordance with these methods include cancers.Polynucleotides corresponding to genes that exhibit the appropriateexpression pattern can be used to detect cancer in a subject. For areview of markers of cancer, see, e.g., Hanahan et al. Cell 100:57-70(2000).

One detection/diagnostic method comprises: (a) obtaining from a mammal(e.g., a human) a biological sample, (b) detecting the presence in thesample of a CA protein and (c) comparing the amount of product presentwith that in a control sample. In accordance with this method, thepresence in the sample of elevated levels of a CA gene product indicatesthat the subject has a neoplastic or preneoplastic condition.

Biological samples suitable for use in this method include biologicalfluids such as serum, plasma, pleural effusions, urine andcerebro-spinal fluid, CSF, tissue samples (e.g., mammary tumor orprostate tissue slices) can also be used in the method of the invention,including samples derived from biopsies. Cell cultures or cell extractsderived, for example, from tissue biopsies can also be used.

The compound is preferably a binding protein, e.g., an antibody,polyclonal or monoclonal, or antigen binding fragment thereof, which canbe labeled with a detectable marker (e.g., fluorophore, chromophore orisotope, etc). Where appropriate, the compound can be attached to asolid support such as a bead, plate, filter, resin, etc. Determinationof formation of the complex can be effected by contacting the complexwith a further compound (e.g., an antibody) that specifically binds tothe first compound (or complex). Like the first compound, the furthercompound can be attached to a solid support and/or can be labeled with adetectable marker.

The identification of elevated levels of CA protein in accordance withthe present invention makes possible the identification of subjectspatients) that are likely to benefit from adjuvant therapy. For example,a biological sample from a post primary therapy subject (e.g., subjecthaving undergone surgery) can be screened for the presence ofcirculating CA protein, the presence of elevated levels of the protein,determined by studies of normal populations, being indicative ofresidual tumor tissue. Similarly, tissue from the cut site of asurgically removed tumor can be examined (e.g., by immunofluorescence),the presence of elevated levels of product (relative to the surroundingtissue) being indicative of incomplete removal of the tumor. The abilityto identify such subjects makes it possible to tailor therapy to theneeds of the particular subject. Subjects undergoing non-surgicaltherapy, e.g., chemotherapy or radiation therapy, can also be monitored,the presence in samples from such subjects of elevated levels of CAprotein being indicative of the need for continued treatment. Staging ofthe disease (for example, for purposes of optimizing treatment regimens)can also be effected, for example, by biopsy e.g., with antibodyspecific for a CA protein.

(g) Animal Models and Transgenics

In another preferred embodiment CA genes find use in generating animalmodels of cancers, particularly lymphomas and carcinomas. As isappreciated by one of ordinary skill in the art, when the CA geneidentified is repressed or diminished in CA tissue, gene therapytechnology wherein antisense RNA directed to the CA gene will alsodiminish or repress expression of the gene. An animal generated as suchserves as an animal model of CA that finds use in screening bioactivedrug candidates. Similarly, gene knockout technology, for example as aresult of homologous recombination with an appropriate gene targetingvector, will result in the absence of the CA protein. When desired,tissue-specific expression or knockout of the CA protein may benecessary.

It is also possible that the CA protein is overexpressed in cancer. Assuch, transgenic animals can be generated that overexpress the CAprotein. Depending on the desired expression level, promoters of variousstrengths can be employed to express the transgene. Also, the number ofcopies of the integrated transgene can be determined and compared for adetermination of the expression level of the transgene. Animalsgenerated by such methods find use as animal models of CA and areadditionally useful in screening for bioactive molecules to treatcancer.

Characterization of CA Sequences

The CA nucleic acid sequences of the invention are depicted in Tables1-16. The sequences in each Table include genomic DNA sequence (mousegenomic sequences mDxx-yyy; human genomic sequences hDxx-yyy), sequencecorresponding to the mRNA(s) generated therefrom (mRxx-yyy; hRxx-yyy)and amino acid sequences of the proteins (mPxx-yyy; hPxx-yyy) encoded bythe mRNA for both mouse and human genes. N/A indicates a gene that hasbeen identified, but for which there has not been a name ascribed.

The mouse and human genomic DNA sequence, sequence corresponding to themRNA(s) generated therefrom and amino acid sequences of the proteins asshown in Tables 1-16 are described according to SEQ ID NOS as follows inTable 17.

TABLE 17 DESIGNATION SEQ ID NO TYPE OF SEQUENCE mD24-004 SEQ ID NO: 1MOUSE GENOMIC SEQUENCE mR24-004.1 SEQ ID NO: 2 MOUSE mRNA SEQUENCEmP24-004.1 SEQ ID NO: 3 MOUSE PROTEIN SEQUENCE hD24-004 SEQ ID NO: 4HUMAN GENOMIC SEQUENCE hR24-004.1 SEQ ID NO: 5 HUMAN mRNA SEQUENCEhP24-004.1 SEQ ID NO: 6 HUMAN PROTEIN SEQUENCE mD24-006 SEQ ID NO: 7MOUSE GENOMIC SEQUENCE mR24-006.1 SEQ ID NO: 8 MOUSE mRNA SEQUENCEmP24-006.1 SEQ ID NO: 9 MOUSE PROTEIN SEQUENCE hD24-006 SEQ ID NO: 10HUMAN GENOMIC SEQUENCE hR24-006.1 SEQ ID NO: 11 HUMAN mRNA SEQUENCEhP24-006.1 SEQ ID NO: 12 HUMAN PROTEIN SEQUENCE hR24-006.2 SEQ ID NO: 13HUMAN mRNA SEQUENCE hP24-006.2 SEQ ID NO: 14 HUMAN PROTEIN SEQUENCEmD24-009 SEQ ID NO: 15 MOUSE GENOMIC SEQUENCE mR24-009.1 SEQ ID NO: 16MOUSE mRNA SEQUENCE mP24-009.1 SEQ ID NO: 17 MOUSE PROTEIN SEQUENCEhD24-009 SEQ ID NO: 18 HUMAN GENOMIC SEQUENCE hR24-009.1 SEQ ID NO: 19HUMAN mRNA SEQUENCE hP24-009.1 SEQ ID NO: 20 HUMAN PROTEIN SEQUENCEmD25-006 SEQ ID NO: 21 MOUSE GENOMIC SEQUENCE mR25-006.1 SEQ ID NO: 22MOUSE mRNA SEQUENCE mP25-006.1 SEQ ID NO: 23 MOUSE PROTEIN SEQUENCEmR25-006.2 SEQ ID NO: 24 MOUSE mRNA SEQUENCE mP25-006.2 SEQ ID NO: 25MOUSE PROTEIN SEQUENCE hD25-006 SEQ ID NO: 26 HUMAN GENOMIC SEQUENCEhR25-006.1 SEQ ID NO: 27 HUMAN mRNA SEQUENCE hP25-006.1 SEQ ID NO: 28HUMAN PROTEIN SEQUENCE hR25-006.2 SEQ ID NO: 29 HUMAN mRNA SEQUENCEhP25-006.2 SEQ ID NO: 30 HUMAN PROTEIN SEQUENCE mD25-007 SEQ ID NO: 31MOUSE GENOMIC SEQUENCE mR25-007.1 SEQ ID NO: 32 MOUSE mRNA SEQUENCEmP25-007.1 SEQ ID NO: 33 MOUSE PROTEIN SEQUENCE hD25-007 SEQ ID NO: 34HUMAN GENOMIC SEQUENCE hR25-007.1 SEQ ID NO: 35 HUMAN mRNA SEQUENCEhP25-007.1 SEQ ID NO: 36 HUMAN PROTEIN SEQUENCE hR25-007.2 SEQ ID NO: 37HUMAN mRNA SEQUENCE hP25-007.2 SEQ ID NO: 38 HUMAN PROTEIN SEQUENCEhR25-007.3 SEQ ID NO: 39 HUMAN mRNA SEQUENCE hP25-007.3 SEQ ID NO: 40HUMAN PROTEIN SEQUENCE mD25-008 SEQ ID NO: 41 MOUSE GENOMIC SEQUENCEmR25-008.1 SEQ ID NO: 42 MOUSE mRNA SEQUENCE mP25-008.1 SEQ ID NO: 43MOUSE PROTEIN SEQUENCE hD25-008 SEQ ID NO: 44 HUMAN GENOMIC SEQUENCEhR25-008.1 SEQ ID NO: 45 HUMAN mRNA SEQUENCE hP25-008.1 SEQ ID NO: 46HUMAN PROTEIN SEQUENCE mD25-009 SEQ ID NO: 47 MOUSE GENOMIC SEQUENCEmR25-009.1 SEQ ID NO: 48 MOUSE mRNA SEQUENCE mP25-009.1 SEQ ID NO: 49MOUSE PROTEIN SEQUENCE hD25-009 SEQ ID NO: 50 HUMAN GENOMIC SEQUENCEhR25-009.1 SEQ ID NO: 51 HUMAN mRNA SEQUENCE hP25-009.1 SEQ ID NO: 52HUMAN PROTEIN SEQUENCE hR25-009.2 SEQ ID NO: 53 HUMAN mRNA SEQUENCEhP25-009.2 SEQ ID NO: 54 HUMAN PROTEIN SEQUENCE hR25-009.3 SEQ ID NO: 55HUMAN mRNA SEQUENCE hP25-009.3 SEQ ID NO: 56 HUMAN PROTEIN SEQUENCEmD25-013 SEQ ID NO: 57 MOUSE GENOMIC SEQUENCE mR25-013.1 SEQ ID NO: 58MOUSE mRNA SEQUENCE mP25-013.1 SEQ ID NO: 59 MOUSE PROTEIN SEQUENCEhD25-013 SEQ ID NO: 60 HUMAN GENOMIC SEQUENCE hR25-013.1 SEQ ID NO: 61HUMAN mRNA SEQUENCE hP25-013.1 SEQ ID NO: 62 HUMAN PROTEIN SEQUENCEhR25-013.2 SEQ ID NO: 63 HUMAN mRNA SEQUENCE hP25-013.2 SEQ ID NO: 64HUMAN PROTEIN SEQUENCE hR25-013.3 SEQ ID NO: 65 HUMAN mRNA SEQUENCEhP25-013.3 SEQ ID NO: 66 HUMAN PROTEIN SEQUENCE hR25-013.4 SEQ ID NO: 67HUMAN mRNA SEQUENCE hP25-013.4 SEQ ID NO: 68 HUMAN PROTEIN SEQUENCEhR25-013.5 SEQ ID NO: 69 HUMAN mRNA SEQUENCE hP25-013.5 SEQ ID NO: 70HUMAN PROTEIN SEQUENCE hR25-013.6 SEQ ID NO: 71 HUMAN mRNA SEQUENCEhP25-013.6 SEQ ID NO: 72 HUMAN PROTEIN SEQUENCE hR25-013.7 SEQ ID NO: 73HUMAN mRNA SEQUENCE hP25-013.7 SEQ ID NO: 74 HUMAN PROTEIN SEQUENCEhR25-013.8 SEQ ID NO: 75 HUMAN mRNA SEQUENCE hP25-013.8 SEQ ID NO: 76HUMAN PROTEIN SEQUENCE mD25-015 SEQ ID NO: 77 MOUSE GENOMIC SEQUENCEmR25-015.1 SEQ ID NO: 78 MOUSE mRNA SEQUENCE mP25-015.1 SEQ ID NO: 79MOUSE PROTEIN SEQUENCE mR25-015.2 SEQ ID NO: 80 MOUSE mRNA SEQUENCEmP25-015.2 SEQ ID NO: 81 MOUSE PROTEIN SEQUENCE hD25-015 SEQ ID NO: 82HUMAN GENOMIC SEQUENCE hR25-015.1 SEQ ID NO: 83 HUMAN mRNA SEQUENCEhP25-015.1 SEQ ID NO: 84 HUMAN PROTEIN SEQUENCE mD25-017 SEQ ID NO: 85MOUSE GENOMIC SEQUENCE mR25-017.1 SEQ ID NO: 86 MOUSE mRNA SEQUENCEmP25-017.1 SEQ ID NO: 87 MOUSE PROTEIN SEQUENCE hD25-017 SEQ ID NO: 88HUMAN GENOMIC SEQUENCE hR25-017.1 SEQ ID NO: 89 HUMAN mRNA SEQUENCEhP25-017.1 SEQ ID NO: 90 HUMAN PROTEIN SEQUENCE hR25-017.2 SEQ ID NO: 91HUMAN mRNA SEQUENCE hP25-017.2 SEQ ID NO: 92 HUMAN PROTEIN SEQUENCEhR25-017.3 SEQ ID NO: 93 HUMAN mRNA SEQUENCE hP25-017.3 SEQ ID NO: 94HUMAN PROTEIN SEQUENCE hR25-017.4 SEQ ID NO: 95 HUMAN mRNA SEQUENCEhP25-017.4 SEQ ID NO: 96 HUMAN PROTEIN SEQUENCE hR25-017.5 SEQ ID NO: 97HUMAN mRNA SEQUENCE hP25-017.5 SEQ ID NO: 98 HUMAN PROTEIN SEQUENCEhR25-017.6 SEQ ID NO: 99 HUMAN mRNA SEQUENCE hP25-017.6 SEQ ID NO: 100HUMAN PROTEIN SEQUENCE hR25-017.7 SEQ ID NO: 101 HUMAN mRNA SEQUENCEhP25-017.7 SEQ ID NO: 102 HUMAN PROTEIN SEQUENCE hR25-017.8 SEQ ID NO:103 HUMAN mRNA SEQUENCE hP25-017.8 SEQ ID NO: 104 HUMAN PROTEIN SEQUENCEhR25-017.9 SEQ ID NO: 105 HUMAN mRNA SEQUENCE hP25-017.9 SEQ ID NO: 106HUMAN PROTEIN SEQUENCE hR25-017.10 SEQ ID NO: 107 HUMAN mRNA SEQUENCEhP25-017.10 SEQ ID NO: 108 HUMAN PROTEIN SEQUENCE mD25-025 SEQ ID NO:109 MOUSE GENOMIC SEQUENCE mR25-025.1 SEQ ID NO: 110 MOUSE mRNA SEQUENCEmP25-025.1 SEQ ID NO: 111 MOUSE PROTEIN SEQUENCE hD25-025 SEQ ID NO: 112HUMAN GENOMIC SEQUENCE hR25-025.1 SEQ ID NO: 113 HUMAN mRNA SEQUENCEhP25-025.1 SEQ ID NO: 114 HUMAN PROTEIN SEQUENCE hR25-025.2 SEQ ID NO:115 HUMAN mRNA SEQUENCE hP25-025.2 SEQ ID NO: 116 HUMAN PROTEIN SEQUENCEhR25-025.3 SEQ ID NO: 117 HUMAN mRNA SEQUENCE hP25-025.3 SEQ ID NO: 118HUMAN PROTEIN SEQUENCE mD26-002 SEQ ID NO: 119 MOUSE GENOMIC SEQUENCEmR26-002.1 SEQ ID NO: 120 MOUSE mRNA SEQUENCE mP26-002.1 SEQ ID NO: 121MOUSE PROTEIN SEQUENCE hD26-002 SEQ ID NO: 122 HUMAN GENOMIC SEQUENCEhR26-002.1 SEQ ID NO: 123 HUMAN mRNA SEQUENCE hP26-002.1 SEQ ID NO: 124HUMAN PROTEIN SEQUENCE mD26-004 SEQ ID NO: 125 MOUSE GENOMIC SEQUENCEmR26-004.1 SEQ ID NO: 126 MOUSE mRNA SEQUENCE mP26-004.1 SEQ ID NO: 127MOUSE PROTEIN SEQUENCE hD26-004 SEQ ID NO: 128 HUMAN GENOMIC SEQUENCEhR26-004.1 SEQ ID NO: 129 HUMAN mRNA SEQUENCE hP26-004.1 SEQ ID NO: 130HUMAN PROTEIN SEQUENCE mD26-005 SEQ ID NO: 131 MOUSE GENOMIC SEQUENCEmR26-005.1 SEQ ID NO: 132 MOUSE mRNA SEQUENCE mP26-005.1 SEQ ID NO: 133MOUSE PROTEIN SEQUENCE mR26-005.2 SEQ ID NO: 134 MOUSE mRNA SEQUENCEmP26-005.2 SEQ ID NO: 135 MOUSE PROTEIN SEQUENCE hD26-005 SEQ ID NO: 136HUMAN GENOMIC SEQUENCE hR26-005.1 SEQ ID NO: 137 HUMAN mRNA SEQUENCEhP26-005.1 SEQ ID NO: 138 HUMAN PROTEIN SEQUENCE hR26-005.2 SEQ ID NO:139 HUMAN mRNA SEQUENCE hP26-005.2 SEQ ID NO: 140 HUMAN PROTEIN SEQUENCEhR26-005.3 SEQ ID NO: 141 HUMAN mRNA SEQUENCE hP26-005.3 SEQ ID NO: 142HUMAN PROTEIN SEQUENCE hR26-005.4 SEQ ID NO: 143 HUMAN mRNA SEQUENCEhP26-005.4 SEQ ID NO: 144 HUMAN PROTEIN SEQUENCE hR26-005.5 SEQ ID NO:145 HUMAN mRNA SEQUENCE hP26-005.5 SEQ ID NO: 146 HUMAN PROTEIN SEQUENCEhR26-005.6 SEQ ID NO: 147 HUMAN mRNA SEQUENCE hP26-005.6 SEQ ID NO: 148HUMAN PROTEIN SEQUENCE hR26-005.7 SEQ ID NO: 149 HUMAN mRNA SEQUENCEhP26-005.7 SEQ ID NO: 150 HUMAN PROTEIN SEQUENCE mD26-009 SEQ ID NO: 151MOUSE GENOMIC SEQUENCE mR26-009.1 SEQ ID NO: 152 MOUSE mRNA SEQUENCEmP26-009.1 SEQ ID NO: 153 MOUSE PROTEIN SEQUENCE hD26-009 SEQ ID NO: 154HUMAN GENOMIC SEQUENCE hR26-009.1 SEQ ID NO: 155 HUMAN mRNA SEQUENCEhP26-009.1 SEQ ID NO: 156 HUMAN PROTEIN SEQUENCE hR26-009.2 SEQ ID NO:157 HUMAN mRNA SEQUENCE hP26-009.2 SEQ ID NO: 158 HUMAN PROTEIN SEQUENCEhR26-009.3 SEQ ID NO: 159 HUMAN mRNA SEQUENCE hP26-009.3 SEQ ID NO: 160HUMAN PROTEIN SEQUENCE hR26-009.4 SEQ ID NO: 161 HUMAN mRNA SEQUENCEhP26-009.4 SEQ ID NO: 162 HUMAN PROTEIN SEQUENCE hR26-009.5 SEQ ID NO:163 HUMAN mRNA SEQUENCE hP26-009.5 SEQ ID NO: 164 HUMAN PROTEIN SEQUENCEhR26-009.6 SEQ ID NO: 165 HUMAN mRNA SEQUENCE hP26-009.6 SEQ ID NO: 166HUMAN PROTEIN SEQUENCE mD26-013 SEQ ID NO: 167 MOUSE GENOMIC SEQUENCEmR26-013.1 SEQ ID NO: 168 MOUSE mRNA SEQUENCE mP26-013.1 SEQ ID NO: 169MOUSE PROTEIN SEQUENCE hD26-013 SEQ ID NO: 170 HUMAN GENOMIC SEQUENCEhR26-013.1 SEQ ID NO: 171 HUMAN mRNA SEQUENCE hP26-013.1 SEQ ID NO: 172HUMAN PROTEIN SEQUENCE hR26-013.2 SEQ ID NO: 173 HUMAN mRNA SEQUENCEhP26-013.2 SEQ ID NO: 174 HUMAN PROTEIN SEQUENCE

The CA sequences were analyzed by Panther™ (Molecular Diagnostics, PaloAlto, Calif.) software designed to detect homologs and enable predictionof molecular function through a system for protein functionalclassification. Human Gene Ontlogy annotations were prepared inaccordance with the Gene Ontology Consortium (Gene Ontology: tool forthe unification of biology. The Gene Ontology Consortium Nature Genet.25: 25-29 (2000)). Similar analysis was carried out by determining IPRinformation regarding the CA polypeptides from InterPro, which is anintegrated documentation resource for protein families, domains andfunctional sites (Apweiler at al. Bioinformatics 16(12): 1145-1150(2000)).

The CA sequences may be classified according to the following predictedgeneral classifications of function by Panther™ analysis, human geneontology and IPR domain information for polypeptides having SEQ ID NOS:6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70,72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116,118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162,164, 166, 172, and 174 as shown in Tables 1-16. The classifications areshown in Table 18 below.

TABLE 18 Human Protein SEQ ID NO: FUNCTION hP24-004.1 SEQ ID NO: 6 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) TRANSFORMING GROWTH FACTORBETA-RELATED (Unassigned) BIOLOGICAL PROCESS Biological processunclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecular functionunclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESStransmembrane receptor protein serine/threonine kinase signalingpathway > TGFbeta receptor signaling pathway oocyte construction > axisdetermination cell communication > cell-cell signaling GO biologicalprocess > developmental processes MOLECULAR FUNCTION ligand binding orcarrier > protein binding CELL COMPONENT GO cellular component >extracellular HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR003942(TGFBETA4) IPR001839 (TGFB) IPR001111 (TGFb propeptide) IPR001839(TGF-beta) IPR001839 (TGF BETA 2) IPR001839 (sp Q64280 TGF4 MOUSE)hP24-006.1 SEQ ID NO: 12 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) TRANSFORMING GROWTH FACTOR BETA-RELATED (TRANSFORMING GROWTHFACTOR BETA 2) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell surface receptor mediated signal transduction (2.11.01.00.00) >Receptor protein serine/ threonine kinase signaling pathway(2.11.01.04.00) MOLECULAR FUNCTIONS Signaling molecule (1.02.00.00.00) >Cytokine (1.02.01.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS cellcommunication > cell-cell signaling cell growth and maintenance > cellproliferation mesoderm development > lymph gland development celldeath > necrosis muscle development > myogenesis MOLECULAR FUNCTION GOmolecular function > cell cycle regulator ligand binding or carrier >protein binding CELL COMPONENT GO cellular component > extracellularextracellular > extracellular space HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR003940 (TGFBETA2) IPR003911 (TGFBETA) IPR001839 (TGFB)IPR001111 (TGFb propeptide) IPR001839 (TGF-beta) IPR001839 (TGF BETA 2)IPR001839 (sp P09858 TGF2 PIG) hP24-006.2 SEQ ID NO: 14 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) TRANSFORMING GROWTH FACTORBETA-RELATED (TRANSFORMING GROWTH FACTOR BETA 2) BIOLOGICAL PROCESSSignal transduction (2.11.00.00.00) > Cell surface receptor mediatedsignal transduction (2.11.01.00.00) > Receptor protein serine/threoninekinase signaling pathway (2.11.01.04.00) MOLECULAR FUNCTIONS Signalingmolecule (1.02.00.00.00) > Cytokine (1.02.01.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS cell communication > cell-cell signaling cell growthand maintenance > cell proliferation cell death > necrosis mesodermdevelopment > lymph gland development muscle development > myogenesisMOLECULAR FUNCTION GO molecular function > cell cycle regulator ligandbinding or carrier > protein binding CELL COMPONENT GO cellularcomponent > extracellular extracellular > extracellular space HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) IPR003940 (TGFBETA2) IPR003911(TGFBETA) IPR001839 (TGFB) IPR001111 (TGFb propeptide) IPR001839(TGF-beta) IPR001839 (TGF BETA 2) IPR001839 (sp P09858 TGF2 PIG)hP24-009.1 SEQ ID NO: 20 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) PLEXIN DOMAIN-CONTAINING RECEPTOR (PLEXIN) HUMAN GENEONTOLOGY BIOLOGICAL PROCESS cell communication > signal transductionMOLECULAR FUNCTION glucosidase > mannosyl-oligosaccharide glucosidase(processing A-glucosidase I) CELL COMPONENT cell > membrane fractionHUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR002909 (IPT) IPR003659(PSI) IPR001627 (Sema) IPR002909 (TIG) IPR002165 (PSI) hP25-006.1 SEQ IDNO: 28 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY)VOLTAGE-DEPENDENT CALCIUM CHANNEL BETA SUBUNIT (VOLTAGE- DEPENDENTCALCIUM CHANNEL BETA-4) BIOLOGICAL PROCESS Transport (2.15.00.00.00) >Ion transport (2.15.01.00.00) > Cation transport (2.15.01.01.00)MOLECULAR FUNCTIONS Ion channel (1.03.00.00.00) > Voltage-gated ionchannel (1.03.03.00.00) > Voltage-gated calcium channel (1.03.03.02.00)HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS di-, tri-valent inorganic cationtransport > calcium ion transport transport > ion transport muscledevelopment > neuromuscular junction development cell motility > musclecontraction peptidoglycan catabolism > mRNA splicing transcription fromPol II promoter > mRNA splicing MOLECULAR FUNCTION RNA dependentadenosinetriphosphatase > ATP dependent RNA helicase ATP dependenthelicase > ATP dependent RNA helicase RNA helicase > ATP dependent RNAhelicase CELL COMPONENT cell > membrane fraction nucleus > spliceosomeintegral plasma membrane protein > voltage-gated calcium channel HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) IPR000584 (LCACHANNELB) IPR000619(GuKc) IPR001452 (SH3) IPR000584 (Ca channel B) IPR001452 (SH3)hP25-006.2 SEQ ID NO: 30 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) VOLTAGE-DEPENDENT CALCIUM CHANNEL BETA SUBUNIT (VOLTAGE-DEPENDENT CALCIUM CHANNEL BETA-4) BIOLOGICAL PROCESS Transport(2.15.00.00.00) > Ion transport (2.15.01.00.00) > Cation transport(2.15.01.01.00) MOLECULAR FUNCTIONS Ion channel (1.03.00.00.00) >Voltage-gated ion channel (1.03.03.00.00) > Voltage-gated calciumchannel (1.03.03.02.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS di-,tri-valent inorganic cation transport > calcium ion transportpeptidoglycan catabolism > mRNA splicing transcription from Pol IIpromoter > mRNA splicing cell motility > muscle contraction muscledevelopment > neuromuscular junction development transport > iontransport MOLECULAR FUNCTION RNA dependent adenosinetriphosphatase > ATPdependent RNA helicase ATP dependent helicase > ATP dependent RNAhelicase RNA helicase > ATP dependent RNA helicase CELL COMPONENT cell >membrane fraction nucleus > spliceosome integral plasma membraneprotein > voltage-gated calcium channel HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR000584 (LCACHANNELB) IPR000619 (GuKc) IPR001452 (SH3)IPR000584 (Ca channel B) IPR001452 (SH3) hP25-007.1 SEQ ID NO: 36 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) SECRETORY PHOSPHOLIPASE A2RECEPTOR-RELATED (SECRETORY PHOSPHOLIPASE A2 RECEPTOR) BIOLOGICALPROCESS Biological process unclassified (2.99.00.00.00) MOLECULARFUNCTIONS Receptor (1.01.00.00.00) > Other receptor (1.01.99.00.00)HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defence response > inflammatoryresponse macromolecule catabolism > proteolysis and peptidolysis cellcommunication > cell adhesion MOLECULAR FUNCTION metalloendopeptidase >gelatinase A peptidase > metallopeptidase metalloendopeptidase >gelatinase B collagenase > interstitial collagenase collagenase >neutrophil collagenase CELL COMPONENT cell > membrane fraction plasmamembrane > integral plasma membrane protein extracellular >extracellular space extracellular > extracellular matrix HUMAN PROTEINDOMAINS (INTERPRO SIGNATURES) IPR000562 (FNTYPEII) IPR001304 (CLECT)IPR000562 (FN2) IPR000772 (RICIN) IPR000562 (fn2) IPR001304 (lectin c)IPR000772 (Ricin B lectin) IPR001304 (C TYPE LECTIN 2 8) IPR000772(RICIN B LECTIN) IPR000562 (sp Q13018 Q13018 HUMAN) hP25-007.2 SEQ IDNO: 38 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) SECRETORYPHOSPHOLIPASE A2 RECEPTOR-RELATED (SECRETORY PHOSPHOLIPASE A2 RECEPTOR)BIOLOGICAL PROCESS Biological process unclassified (2.99.00.00.00)MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) > Other receptor(1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response macromolecule catabolism > proteolysisand peptidolysis cell communication > cell adhesion MOLECULAR FUNCTIONmetalloendopeptidase > gelatinase A peptidase > metallopeptidasemetalloendopeptidase > gelatinase B collagenase > interstitialcollagenase collagenase > neutrophil collagenase CELL COMPONENT cell >membrane fraction plasma membrane > integral plasma membrane proteinextracellular > extracellular space extracellular > extracellular matrixHUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR000562 (FNTYPEII)IPR001304 (CLECT) IPR000562 (FN2) IPR000772 (RICIN) IPR000562 (fn2)IPR001304 (lectin c) IPR000772 (Ricin B lectin) IPR001304 (C TYPE LECTIN2 8) IPR000772 (RICIN B LECTIN) IPR000562 (sp Q13018 Q13018 HUMAN)hP25-007.3 SEQ ID NO: 40 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) SECRETORY PHOSPHOLIPASE A2 RECEPTOR-RELATED (SECRETORYPHOSPHOLIPASE A2 RECEPTOR) BIOLOGICAL PROCESS Biological processunclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Receptor(1.01.00.00.00) > Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS defence response > inflammatory responsemacromolecule catabolism > proteolysis and peptidolysis cellcommunication > cell adhesion MOLECULAR FUNCTION metalloendopeptidase >gelatinase A peptidase > metallopeptidase metalloendopeptidase >gelatinase B collagenase > interstitial collagenase collagenase >neutrophil collagenase CELL COMPONENT cell > membrane fraction plasmamembrane > integral plasma membrane protein extracellular >extracellular space extracellular > extracellular matrix HUMAN PROTEINDOMAINS (INTERPRO SIGNATURES) IPR000562 (FNTYPEII) IPR001304 (CLECT)IPR000562 (FN2) IPR000772 (RICIN) IPR000562 (fn2) IPR001304 (lectin c)IPR000772 (Ricin B lectin) IPR001304 (C TYPE LECTIN 2 8) IPR000772(RICIN B LECTIN) IPR000562 (sp Q13018 Q13018 HUMAN) hP25-008.1 SEQ IDNO: 46 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) AMINO ACIDTRANSPORTER- RELATED (gb def: (ae003835) cg13743 gene product[drosophila melanogaster]) BIOLOGICAL PROCESS Amino acid metabolism(2.02.00.00.00) Transport (2.15.00.00.00) > Amino acid transport(2.15.05.00.00) MOLECULAR FUNCTIONS Transporter (1.10.00.00.00) > Othertransporter (1.10.99.00.00) HUMAN GENE ONTOLOGY No Gene Ontology HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) IPR002422 (AROMATIC AA PERM 2)hP25-009.1 SEQ ID NO: 52 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) MITOCHONDRIAL CARRIER PROTEIN (MITOCHONDRIAL CARRIERPROTEIN) BIOLOGICAL PROCESS Transport (2.15.00.00.00) > Small moleculetransport (2.15.02.00.00) MOLECULAR FUNCTIONS Transfer/carrier protein(1.11.00.00.00) > Mitochondrial carrier protein (1.11.02.00.00) HUMANGENE ONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR002048 (EFh) IPR001993 (mito carr) IPR002048 (efhand)IPR002048 (EF HAND 2) hP25-009.2 SEQ ID NO: 54 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) MITOCHONDRIAL CARRIER PROTEIN(MITOCHONDRIAL CARRIER PROTEIN) BIOLOGICAL PROCESS Transport(2.15.00.00.00) > Small molecule transport (2.15.02.00.00) MOLECULARFUNCTIONS Transfer/carrier protein (1.11.00.00.00) > Mitochondrialcarrier protein (1.11.02.00.00) HUMAN GENE ONTOLOGY No Gene OntologyHUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR001993 (mito carr)hP25-009.3 SEQ ID NO: 56 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) MITOCHONDRIAL CARRIER PROTEIN (MITOCHONDRIAL CARRIERPROTEIN) BIOLOGICAL PROCESS Transport (2.15.00.00.00) > Small moleculetransport (2.15.02.00.00) MOLECULAR FUNCTIONS Transfer/carrier protein(1.11.00.00.00) > Mitochondrial carrier protein (1.11.02.00.00) HUMANGENE ONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR002048 (EFh) IPR001993 (mito carr) IPR002048 (efhand)IPR002048 (EF HAND 2) hP25-013.1 SEQ ID NO: 62 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE (EMBRYONICEPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological process unknown(2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response MOLECULAR FUNCTION sugar binding >lectin CELL COMPONENT nuclear membrane > nuclear membrane lumen plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) No Domain Hit hP25-013.2 SEQ ID NO: 64 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE(EMBRYONIC EPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological processunknown (2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response MOLECULAR FUNCTION sugar binding >lectin CELL COMPONENT nuclear membrane > nuclear membrane lumen plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) No Domain Hit hP25-013.3 SEQ ID NO: 66 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE(EMBRYONIC EPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological processunknown (2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response MOLECULAR FUNCTION sugar binding >lectin CELL COMPONENT nuclear membrane > nuclear membrane lumen plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) No Domain Hit hP25-013.4 SEQ ID NO: 68 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE(EMBRYONIC EPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological processunknown (2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response MOLECULAR FUNCTION sugar binding >lectin CELL COMPONENT nuclear membrane > nuclear membrane lumen plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) No Domain Hit hP25-013.5 SEQ ID NO: 70 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED)BIOLOGICAL PROCESS Biological process unclassified (2.99.00.00.00)MOLECULAR FUNCTIONS Molecular function unclassified (1.97.00.00.00)HUMAN GENE ONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) No Domain Hit hP25-013.6 SEQ ID NO: 72 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE (EMBRYONICEPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological process unknown(2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response MOLECULAR FUNCTION sugar binding >lectin CELL COMPONENT nuclear membrane > nuclear membrane lumen plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) No Domain Hit hP25-013.7 SEQ ID NO: 74 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CANCER OVEREXPRESSED GENE(EMBRYONIC EPITHELIA PROTEIN) BIOLOGICAL PROCESS Biological processunknown (2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unknown(1.99.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS defenceresponse > inflammatory response isoprenoid catabolism > nitrogenmetabolism cell death > apoptosis defence response > immune responseMOLECULAR FUNCTION sugar binding > lectin CELL COMPONENT plasmamembrane > integral plasma membrane protein GO cellular component >extracellular HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR002352(EMAJORBASICP) IPR001304 (CLECT) IPR001304 (lectin c) IPR001304 (C TYPELECTIN 2) hP25-013.8 SEQ ID NO: 76 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) CANCER OVEREXPRESSED GENE (EMBRYONIC EPITHELIA PROTEIN)BIOLOGICAL PROCESS Biological process unknown (2.97.00.00.00) MOLECULARFUNCTIONS Molecular function unknown (1.99.00.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS defence response > inflammatory response isoprenoidcatabolism > nitrogen metabolism cell death > apoptosis defenceresponse > immune response MOLECULAR FUNCTION sugar binding > lectinCELL COMPONENT plasma membrane > integral plasma membrane protein GOcellular component > extracellular HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR002352 (EMAJORBASICP) IPR001304 (CLECT) IPR001304 (lectinc) IPR001304 (C TYPE LECTIN 2) hP25-015.1 SEQ ID NO: 84 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) LEUCINE-RICH REPEAT PROTEIN- RELATED(CHONDROADHERIN-RELATED) BIOLOGICAL PROCESS Biological process unknown(2.97.00.00.00) MOLECULAR FUNCTIONS Molecular function unclassified(1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS cellcommunication > cell adhesion MOLECULAR FUNCTION GO molecular function >cell adhesion CELL COMPONENT cell > plasma membrane HUMAN PROTEINDOMAINS (INTERPRO SIGNATURES) IPR001611 (LEURICHRPT) IPR003596 (IGv)NULL (LRR PS) IPR003598 (IGc2) IPR000372 (LRRNT) IPR003599 (IG)IPR003591 (LRR TYP) IPR001611 (LRR) IPR000372 (LRRNT) IPR003006 (ig)IPR000483 (LRRCT) NULL (THR RICH) hP25-017.1 SEQ ID NO: 90 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN (CD44 ANTIGEN)BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) > Cellcommunication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.2 SEQ IDNO: 92 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.3 SEQ IDNO: 94 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION transmembrane receptor > cell adhesion receptor celladhesion > cell adhesion receptor glycosaminoglycan binding > hyaluronicacid binding molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.4 SEQ IDNO: 96 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.5 SEQ IDNO: 98 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.6 SEQ IDNO: 100 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION transmembrane receptor > cell adhesion receptor celladhesion > cell adhesion receptor glycosaminoglycan binding > hyaluronicacid binding molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.7 SEQ IDNO: 102 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.8 SEQ IDNO: 104 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION glycosaminoglycan binding > hyaluronic acid bindingtransmembrane receptor > cell adhesion receptor cell adhesion > celladhesion receptor molecular_function unknown > lymphocyte antigen CELLCOMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538 (LINK)IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-017.9 SEQ IDNO: 106 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CD44 ANTIGEN(CD44 ANTIGEN) BIOLOGICAL PROCESS Signal transduction (2.11.00.00.00) >Cell communication (2.11.03.00.00) > Cell adhesion- mediated signaling(2.11.03.01.00) Signal transduction (2.11.00.00.00) > Intracellularsignaling cascade (2.11.02.00.00) > Calcium mediated signaling(2.11.02.02.00) > MAPKKK cascade (2.11.02.03.00) Immunity and defense(2.16.00.00.00) > B- cell- and antibody-mediated immunity(2.16.02.00.00) Immunity and defense (2.16.00.00.00) >Macrophage-mediated immunity (2.16.05.00.00) Immunity and defense(2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00) Oncogenesis(2.17.00.00.00) > Other oncogenesis (2.17.99.00.00) Apoptosis(2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cell structureand motility (2.27.00.00.00) > Cell motility (2.27.02.00.00) Celladhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor (1.01.00.00.00) >Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESScell communication > cell adhesion stress response > defence responseMOLECULAR FUNCTION molecular_function unknown > lymphocyte antigenglycosaminoglycan binding > hyaluronic acid binding transmembranereceptor > cell adhesion receptor cell adhesion > cell adhesion receptorCELL COMPONENT cell > membrane fraction cell > plasma membraneextracellular > extracellular matrix integral plasma membrane protein >integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR000538 (LINK) IPR000538 (Xlink) IPR000538 (sp O95370O95370 HUMAN) hP25-017.10 SEQ ID NO: 108 HUMAN PANTHER CLASSIFICATIONSFAMILY (SUBFAMILY) CD44 ANTIGEN (CD44 ANTIGEN) BIOLOGICAL PROCESS Signaltransduction (2.11.00.00.00) > Cell communication (2.11.03.00.00) > Celladhesion- mediated signaling (2.11.03.01.00) Signal transduction(2.11.00.00.00) > Intracellular signaling cascade (2.11.02.00.00) >Calcium mediated signaling (2.11.02.02.00) > MAPKKK cascade(2.11.02.03.00) Immunity and defense (2.16.00.00.00) > B- cell- andantibody-mediated immunity (2.16.02.00.00) Immunity and defense(2.16.00.00.00) > Macrophage-mediated immunity (2.16.05.00.00) Immunityand defense (2.16.00.00.00) > T- cell mediated immunity (2.16.01.00.00)Oncogenesis (2.17.00.00.00) > Other oncogenesis (2.17.99.00.00)Apoptosis (2.26.00.00.00) > Inhibition of apoptosis (2.26.03.00.00) Cellstructure and motility (2.27.00.00.00) > Cell motility (2.27.02.00.00)Cell adhesion (2.29.00.00.00) MOLECULAR FUNCTIONS Receptor(1.01.00.00.00) > Other receptor (1.01.99.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS cell communication > cell adhesion stress response >defence response MOLECULAR FUNCTION glycosaminoglycan binding >hyaluronic acid binding transmembrane receptor > cell adhesion receptorcell adhesion > cell adhesion receptor molecular_function unknown >lymphocyte antigen CELL COMPONENT cell > membrane fraction cell > plasmamembrane extracellular > extracellular matrix integral plasma membraneprotein > integral plasma membrane proteoglycan HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) IPR001231 (CD44) IPR000538 (LINKMODULE) IPR000538(LINK) IPR000538 (Xlink) IPR000538 (sp Q92493 Q92493 HUMAN) hP25-025.1SEQ ID NO: 114 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY)PHOSPHATE TRANSPORTER- RELATED (PHOSPHATE TRANSPORTER SLC20A1-RELATED)BIOLOGICAL PROCESS Phosphate metabolism (2.09.00.00.00) Transport(2.15.00.00.00) > Phosphate transport (2.15.10.00.00) MOLECULARFUNCTIONS Transporter (1.10.00.00.00) > Other transporter(1.10.99.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS isoprenoidcatabolism > phosphate metabolism cell growth and maintenance >transport inorganic anion transport > phosphate transport MOLECULARFUNCTION double-stranded RNA binding > double- stranded RNA adenosinedeaminase RNA editase > double-stranded RNA adenosine deaminase CELLCOMPONENT cell > membrane fraction plasma membrane > integral plasmamembrane protein HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR001204(PHO4) hP25-025.2 SEQ ID NO: 116 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) Unclassified HUMAN GENE ONTOLOGY No Gene Ontology HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) No Domain Hit hP25-025.3 SEQ IDNO: 118 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) PHOSPHATETRANSPORTER- RELATED (PHOSPHATE TRANSPORTER SLC20A1-RELATED) BIOLOGICALPROCESS Phosphate metabolism (2.09.00.00.00) Transport (2.15.00.00.00) >Phosphate transport (2.15.10.00.00) MOLECULAR FUNCTIONS Transporter(1.10.00.00.00) > Other transporter (1.10.99.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS isoprenoid catabolism > phosphate metabolism cellgrowth and maintenance > transport inorganic anion transport > phosphatetransport MOLECULAR FUNCTION double-stranded RNA binding > double-stranded RNA adenosine deaminase RNA editase > double-stranded RNAadenosine deaminase CELL COMPONENT cell > membrane fraction plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) IPR001204 (PHO4) hP26-002.1 SEQ ID NO: 124 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CF10647 (gb def: (x95283)c-serate-1 protein [gallus gallus]) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICALPROCESS eye morphogenesis > mystery cell fate differentiation GObiological process > developmental processes water-soluble vitaminmetabolism > biotin metabolism cell communication > cell adhesion cellcommunication > cell recognition MOLECULAR FUNCTION ligand binding orcarrier > calcium binding defense/immunity protein > immunoglobulin Bcell receptor > immunoglobulin glycosaminoglycan binding > hyaluronicacid binding GO molecular function > cell adhesion CELL COMPONENT cell >membrane fraction plasma membrane > integral plasma membrane proteinextracellular > extracellular matrix cytoplasm > lipid particle HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) IPR001438 (EGFBLOOD) IPR001774(DSL) IPR001881 (EGF CA) IPR001007 (VWC) IPR000561 (EGF) NULL (VWC out)IPR000561 (EGF) IPR001774 (DSL) IPR001881 (EGF CA 2 10) IPR000742 (EGF 2DOMAIN 11) NULL (CYS RICH) hP26-004.1 SEQ ID NO: 130 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) CF10820 (PROTEIN-TYROSINEPHOSPHATASE) BIOLOGICAL PROCESS Protein metabolism and modification(2.05.00.00.00) > Protein modification (2.05.03.00.00) > Proteinphosphorylation (2.05.03.01.00) MOLECULAR FUNCTIONS Phosphatase(1.16.00.00.00) > Protein phosphatase (1.16.01.00.00) HUMAN GENEONTOLOGY BIOLOGICAL PROCESS protein modification > proteindephosphorylation enzyme linked receptor protein signaling pathway >transmembrane receptor protein tyrosine kinase signaling pathway enzymelinked receptor protein signaling pathway > transmembrane receptorprotein tyrosine phosphatase signaling pathway axon guidance > motoraxon guidance MOLECULAR FUNCTION protein tyrosine phosphatase >transmembrane receptor protein tyrosine phosphatase transmembranereceptor > transmembrane receptor protein tyrosine phosphatase proteinphosphatase > protein tyrosine phosphatase enzyme > protein phosphataseprotein tyrosine phosphatase > prenylated protein tyrosine phosphataseprotein tyrosine phosphatase > non- membrane spanning protein tyrosinephosphatase CELL COMPONENT cell > membrane fraction cytoplasm >cytoskeleton cell > plasma membrane plasma membrane > integral plasmamembrane protein cell > cytoplasm HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR000998 (MAMDOMAIN) IPR003962 (FNTYPEIII) IPR000242(PRTYPHPHTASE) IPR000242 (PTPc) IPR003961 (FN3) IPR000998 (MAM)IPR003595 (PTPc motif) IPR003961 (fn3) IPR000242 (Y phosphatase)IPR000998 (MAM) IPR000387 (TYR PHOSPHATASE 2 2) IPR000998 (MAM 2)IPR000242 (TYR PHOSPHATASE PTP 2) hP26-005.1 SEQ ID NO: 138 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED)BIOLOGICAL PROCESS Biological process unclassified (2.99.00.00.00)MOLECULAR FUNCTIONS Molecular function unclassified (1.97.00.00.00)HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS peptidoglycan catabolism >pheromone induction of gene expression from Pol II promoter pheromoneinduction of gene expression > pheromone induction of gene expressionfrom Pol II promoter transcription regulation from Pol II promoter >pheromone induction of gene expression from Pol II promoter pheromoneresponse > pheromone induction of gene expression from Pol II promoterMOLECULAR FUNCTION enzyme > N- acetylglucosaminylphosphatidylinositoldeacetylase CELL COMPONENT plasma membrane > integral plasma membraneprotein HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) No Domain HithP26-005.2 SEQ ID NO: 140 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICALPROCESS peptidoglycan catabolism > pheromone induction of geneexpression from Pol II promoter pheromone induction of gene expression >pheromone induction of gene expression from Pol II promotertranscription regulation from Pol II promoter > pheromone induction ofgene expression from Pol II promoter pheromone response > pheromoneinduction of gene expression from Pol II promoter MOLECULAR FUNCTIONenzyme > N- acetylglucosaminylphosphatidylinositol deacetylase CELLCOMPONENT plasma membrane > integral plasma membrane protein HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) NULL (LEU RICH) hP26-005.3 SEQ IDNO: 142 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) NOT ANNOTATED(NOT ANNOTATED) BIOLOGICAL PROCESS Biological process unclassified(2.99.00.00.00) MOLECULAR FUNCTIONS Molecular function unclassified(1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS peptidoglycancatabolism > pheromone induction of gene expression from Pol II promoterpheromone induction of gene expression > pheromone induction of geneexpression from Pol II promoter transcription regulation from Pol IIpromoter > pheromone induction of gene expression from Pol II promoterpheromone response > pheromone induction of gene expression from Pol IIpromoter MOLECULAR FUNCTION enzyme > N-acetylglucosaminylphosphatidylinositol deacetylase CELL COMPONENT plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) NULL (LEU RICH) hP26-005.4 SEQ ID NO: 144 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED)BIOLOGICAL PROCESS Biological process unclassified (2.99.00.00.00)MOLECULAR FUNCTIONS Molecular function unclassified (1.97.00.00.00)HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS peptidoglycan catabolism >pheromone induction of gene expression from Pol II promoter pheromoneinduction of gene expression > pheromone induction of gene expressionfrom Pol II promoter transcription regulation from Pol II promoter >pheromone induction of gene expression from Pol II promoter pheromoneresponse > pheromone induction of gene expression from Pol II promoterMOLECULAR FUNCTION enzyme > N- acetylglucosaminylphosphatidylinositoldeacetylase CELL COMPONENT plasma membrane > integral plasma membraneprotein HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) NULL (LEU RICH)hP26-005.5 SEQ ID NO: 146 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY No GeneOntology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) No Domain HithP26-005.6 SEQ ID NO: 148 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) NOT ANNOTATED (NOT ANNOTATED) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICALPROCESS peptidoglycan catabolism > pheromone induction of geneexpression from Pol II promoter pheromone induction of gene expression >pheromone induction of gene expression from Pol II promotertranscription regulation from Pol II promoter > pheromone induction ofgene expression from Pol II promoter pheromone response > pheromoneinduction of gene expression from Pol II promoter MOLECULAR FUNCTIONenzyme > N- acetylglucosaminylphosphatidylinositol deacetylase CELLCOMPONENT plasma membrane > integral plasma membrane protein HUMANPROTEIN DOMAINS (INTERPRO SIGNATURES) NULL (LEU RICH) hP26-005.7 SEQ IDNO: 150 HUMAN PANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) NOT ANNOTATED(NOT ANNOTATED) BIOLOGICAL PROCESS Biological process unclassified(2.99.00.00.00) MOLECULAR FUNCTIONS Molecular function unclassified(1.97.00.00.00) HUMAN GENE ONTOLOGY BIOLOGICAL PROCESS peptidoglycancatabolism > pheromone induction of gene expression from Pol II promoterpheromone induction of gene expression > pheromone induction of geneexpression from Pol II promoter transcription regulation from Pol IIpromoter > pheromone induction of gene expression from Pol II promoterpheromone response > pheromone induction of gene expression from Pol IIpromoter MOLECULAR FUNCTION enzyme > N-acetylglucosaminylphosphatidylinositol deacetylase CELL COMPONENT plasmamembrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) NULL (LEU RICH) hP26-009.1 SEQ ID NO: 156 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) Unclassified HUMAN GENEONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) NoDomain Hit hP26-009.2 SEQ ID NO: 158 HUMAN PANTHER CLASSIFICATIONSFAMILY (SUBFAMILY) CADHERIN (gb def: (al109928) dj551d2.1.1(cadherin-like protein vr20, isoform 1) [homo sapiens]) BIOLOGICALPROCESS Biological process unclassified (2.99.00.00.00) MOLECULARFUNCTIONS Molecular function unclassified (1.97.00.00.00) HUMAN GENEONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) NoDomain Hit hP26-009.3 SEQ ID NO: 160 HUMAN PANTHER CLASSIFICATIONSFAMILY (SUBFAMILY) CADHERIN (gb def: (al109928) dj551d2.1.1(cadherin-like protein vr20, isoform 1) [homo sapiens]) BIOLOGICALPROCESS Biological process unclassified (2.99.00.00.00) MOLECULARFUNCTIONS Molecular function unclassified (1.97.00.00.00) HUMAN GENEONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)IPR002126 (CA) IPR002126 (cadherin) IPR000233 (Cadherin C term)IPR002126 (CADHERIN 2 3) hP26-009.4 SEQ ID NO: 162 HUMAN PANTHERCLASSIFICATIONS FAMILY (SUBFAMILY) CADHERIN (gb def: (al109928)dj551d2.1.1 (cadherin-like protein vr20, isoform 1) [homo sapiens])BIOLOGICAL PROCESS Biological process unclassified (2.99.00.00.00)MOLECULAR FUNCTIONS Molecular function unclassified (1.97.00.00.00)HUMAN GENE ONTOLOGY No Gene Ontology HUMAN PROTEIN DOMAINS (INTERPROSIGNATURES) IPR002126 (CA) IPR002126 (cadherin) IPR002126 (CADHERIN 2 3)hP26-009.5 SEQ ID NO: 164 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) CADHERIN (gb def: (al109928) dj551d2.1.1 (cadherin-likeprotein vr20, isoform 1) [homo sapiens]) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY No GeneOntology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR002126(CADHERIN) IPR002126 (CA) IPR002126 (cadherin) IPR002126 (CADHERIN 2 4)hP26-009.6 SEQ ID NO: 166 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) CADHERIN (gb def: (al109928) dj551d2.1.1 (cadherin-likeprotein vr20, isoform 1) [homo sapiens]) BIOLOGICAL PROCESS Biologicalprocess unclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Molecularfunction unclassified (1.97.00.00.00) HUMAN GENE ONTOLOGY No GeneOntology HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES) IPR002126(CADHERIN) IPR002126 (CA) IPR002126 (cadherin) IPR002126 (CADHERIN 2 4)hP26-013.1 SEQ ID NO: 172 HUMAN PANTHER CLASSIFICATIONS FAMILY(SUBFAMILY) CADHERIN (gb def: (ab046782) kiaa1562 protein [homosapiens]) BIOLOGICAL PROCESS Biological process unclassified(2.99.00.00.00) MOLECULAR FUNCTIONS Cell adhesion molecule(1.05.00.00.00) > Cadherin (1.05.02.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS cell communication > cell adhesion cellcommunication > cell-cell signaling ectoderm development > neurogenesisMOLECULAR FUNCTION cell adhesion > calcium-dependent cell adhesion GOmolecular function > cell adhesion CELL COMPONENT cell > plasma membraneplasma membrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) IPR002126 (CADHERIN) IPR002126 (CA) IPR002126(cadherin) IPR002126 (CADHERIN 2 6) hP26-013.2 SEQ ID NO: 174 HUMANPANTHER CLASSIFICATIONS FAMILY (SUBFAMILY) CADHERIN (gb def: (ab046782)kiaa1562 protein [homo sapiens]) BIOLOGICAL PROCESS Biological processunclassified (2.99.00.00.00) MOLECULAR FUNCTIONS Cell adhesion molecule(1.05.00.00.00) > Cadherin (1.05.02.00.00) HUMAN GENE ONTOLOGYBIOLOGICAL PROCESS cell communication > cell adhesion cellcommunication > cell-cell signaling ectoderm development > neurogenesisMOLECULAR FUNCTION cell adhesion > calcium-dependent cell adhesion GOmolecular function > cell adhesion CELL COMPONENT cell > plasma membraneplasma membrane > integral plasma membrane protein HUMAN PROTEIN DOMAINS(INTERPRO SIGNATURES) IPR002126 (CADHERIN) IPR002126 (CA) IPR002126(cadherin) IPR002126 (CADHERIN 2 6)

A CA protein (CAP) is a signalling protein wherein the CAP sequence isselected from the group consisting of SEQ ID NOS: 6, 12, 14, 96, 130,172 and 174 shown in Tables 1-16.

A CA protein (CAP) is involved in signal transduction wherein the CAPsequence is selected from the group consisting of SEQ ID NOS: 20, 84,90, 92, 94, 96, 98, 100, 102, 104, 106, and 108 shown in Tables 1-16.

A CA protein (CAP) is a cell adhesion protein wherein the CAP sequenceis selected from the group consisting of SEQ ID NOS: 36, 38, 40, 84, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 124, 172, and 174 as shown inTables 1-16.

A CA protein (CAP) is involved in inflammatory response wherein the CAPsequence is selected from the group consisting of SEQ ID NOS: 62, 64,66, 68, 72, 74, and 76 as shown in Tables 1-16.

A CA protein (CAP) is involved in pheromone response wherein the CAPsequence is selected from the group consisting of SEQ ID NOS: 138, 140,142, 144, 146, 148, and 150 as shown in Tables 1-16.

A CA protein (CAP) is an ion transport protein wherein the CAP sequenceis selected from the group consisting of SEQ ID NOS: 28, 30, 114, and118 as shown in Tables 1-16.

A CA protein (CAP) is expressed on a cell surface, wherein the CAprotein is selected from the group consisting of SEQ ID NOS: 6, 12, 14,20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70, 72, 74, 76,84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116, 118, 124,130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162, 164, 166,172, and 174 as shown in Tables 1-16.

Certain aspects of the present invention are described in greater detailin the non-limiting examples that follow.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all and onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Insertion Site Analysis Following Tumor Induction in Mice

Tumors are induced in mice using either mouse mammary tumor virus (MMTV)or murine leukemia virus (MLV). MMTV causes mammary adenocarcinomas andMLV causes a variety of different hematopoetic malignancies (primarilyT- or B-cell lymphomas). Three routes of infection are used: (1)injection of neonates with purified virus preparations, (2) infection bymilk-borne virus during nursing, and (3) genetic transmission ofpathogenic proviruses via the germ-line (Akvr1 and/or Mtv2). The type ofmalignancy present in each affected mouse is determined by histologicalanalysis of H&E-stained thin sections of formalin-fixed,paraffin-embedded biopsy samples. Host DNA sequences flanking allclonally-integrated proviruses in each tumor are recovered by nestedanchored-PCR using two virus-specific primers and two primers specificfor a 40 bp double stranded DNA anchor ligated to restriction enzymedigested tumor DNA. Amplified bands representing host/virus junctionfragments are cloned and sequenced. Then the host sequences (called“tags”) are used to BLAST analyze the Celera mouse genomic sequence. Foreach individual tag, three parameters are recorded: (1) the mousechromosome assignment, (2) base pair coordinates at which theintegration occurred, and (3) provirus orientation. Using thisinformation, all available tags from all analyzed tumors are mapped tothe mouse genome. To identify the protooncogene targets of provirusinsertion mutation, the provirus integration pattern at each cluster ofintegrants is analyzed relative to the locations of all known genes inthe transcriptome. The presence of provirus at the same locus in two ormore independent tumors is prima facie evidence that a protooncogene ispresent at or very near the proviral integration sites. This is becausethe genome is too large for random integrations to result in observableclustering. Any clustering that is detected is unequivocal evidence forbiological selection during tumorigenesis. In order to identify thehuman orthologs of the protooncogene targets of provirus insertionmutation, a comparative analysis of syntenic regions of the mouse andhuman genomes is performed.

An example of PCR amplification of host/virus junction fragments ispresented in FIG. 1. Lane 1 contains the amplification products fromnormal control DNA and lane 2 contains the amplification products fromtumor DNA. The bands result from 5′ host/virus junction fragmentspresent in the DNA samples. Lane 1 has bands from the env/3′ LTRjunctions from all proviruses (upper) and the host/5′ LTR from thepathogenic endogenous Mtv2 provirus present in this particular mousestrain. This endogenous provirus is detected because its sequence isidentical to the new clonally integrated proviruses in the tumor. Allfour new clonally integrated proviruses known to be in this tumor arereadily detected.

Example 2 Analysis of Quantitative RT-PCR: Comparative C_(T) Method

The expression level of target genes is quantified using the ABI PRISM7900HT Sequence Detection System (Applied Biosystems, California). Themethod is based on the quantitation of the initial copy number of targettemplate in comparison to that of a reference (normalizer) housekeepergene (Pre-Developed TaqMan® Assay Reagents Gene ExpressionQuantification Protocol, Applied Biosystems, 2001). Accumulation of DNAproduct with each PCR cycle is related to amplicon efficiency and theinitial template concentration. Therefore the amplification efficiencyof both the target and the normalizer must be approximately equal. Thethreshold cycle (C_(T)), which is dependent on the starting templatecopy number and the DNA amplification efficiency, is a PCR cycle duringwhich PCR product growth is exponential. With a similar dynamic rangefor the target and normalizer, the comparative C_(T) method isapplicable.

An example of the comparative C_(T) method of gene expression forquantitative RT-PCR is shown in FIG. 2. In the first step, assays areperformed in quadruplicate on a normal tissue and several sampletissues. In these tissues, the means and standard deviations of C_(T)values are determined for housekeeper genes (chosen as controls if shownto be biologically stable among various samples, irrespective of diseasestate) and for the target gene. FIG. 2 shows an example of average C_(T)values for a housekeeper gene and target gene. These values can fallwithin a range from upper teens to 40 depending on the intrinsicexpression level of the gene in the particular tissue. The coefficientof variance of all replicate sets cannot exceed 1.5%.

An assessment of how the ΔC_(T) changes with template dilution verifiesthat the efficiencies of the target and housekeeper amplicons areapproximately equal if the log input amount of template RNA versusΔC_(T) plot has a slope <0.10. With the relative efficiencies verifiedfor target and housekeeper, the ΔΔC_(T) comparative calculation becomesvalid, as mentioned above. An example of the calculated differencebetween the C_(T) values of target and housekeeper genes (ΔC_(T)) forvarious samples is shown in FIG. 3. The ΔΔC_(T) is calculated for eachsample by subtracting its ΔC_(T) value from the ΔC_(T) value of thebaseline (calibrator) sample. If the expression is increased in somesamples and decreased in others, ΔΔC_(T) will be a mixture of negativeand positive values. The final step in the calculation is to transformthese values to absolute values. The formula for this is:

Comparative expression level=2^(−ΔΔCT)

The final value for the calibrator should always be one. FIG. 4 showsthe ΔΔC_(T) and comparative expression level for each sample from FIG.3.

Example 3 Detection of Elevated Levels of cDNA Associated with CancerUsing Arrays

cDNA sequences representing a variety of candidate CA genes to bescreened for differential expression in cancer are assayed byhybridization on polynucleotide arrays. The cDNA sequences include cDNAclones isolated from cell lines or tissues of interest. The cDNAsequences analyzed also include polynucleotides comprising sequenceoverlap with sequences in the Unigene database, and which encode avariety of gene products of various origins, functionality, and levelsof characterization. cDNAs are spotted onto reflective slides (Amersham)according to methods well known in the art at a density of 9,216 spotsper slide representing 4,068 sequences (including controls) spotted induplicate, with approximately 0.8 μl of an approximately 200 ng/μlsolution of cDNA.

PCR products of selected cDNA clones corresponding to the gene productsof interest are prepared in a 50% DMSO solution. These PCR products arespotted onto Amersham aluminum microarray slides at a density of 9216clones per array using a Molecular Dynamics Generation III spottingrobot. Clones are spotted in duplicate, for a total of 4608 differentsequences per chip.

cDNA probes are prepared from total RNA obtained by laser capturemicrodissection (LCM, Arcturus Engineering Inc., Mountain View, Calif.)of tumor tissue samples and normal tissue samples isolated frompatients.

Total RNA is first reverse transcribed into cDNA using a primercontaining a T7 RNA polymerase promoter, followed by second strand DNAsynthesis. cDNA is then transcribed in vitro to produce antisense RNAusing the T7 promoter-mediated expression (see, e.g., Luo et al. (1999)Nature Med 5:117-122), and the antisense RNA is then converted intocDNA. The second set of cDNAs are again transcribed in vitro, using theT7 promoter, to provide antisense RNA. This antisense RNA is thenfluorescently labeled, or the RNA is again converted into cDNA, allowingfor a third round of T7-mediated amplification to produce more antisenseRNA. Thus the procedure provides for two or three rounds of in vitrotranscription to produce the final RNA used for fluorescent labeling.Probes are labeled by making fluorescently labeled cDNA from the RNAstarting material. Fluorescently labeled cDNAs prepared from the tumorRNA sample are compared to fluorescently labeled cDNAs prepared fromnormal cell RNA sample. For example, the cDNA probes from the normalcells are labeled with Cy3 fluorescent dye (green) and the cDNA probesprepared from suspected cancer cells are labeled with Cy5 fluorescentdye (red).

The differential expression assay is performed by mixing equal amountsof probes from tumor cells and normal cells of the same patient. Thearrays are prehybridized by incubation for about 2 hrs at 60° C. in5×SSC, 0.2% SDS, 1 mM EDTA, and then washing three times in water andtwice in isopropanol. Following prehybridization of the array, the probemixture is then hybridized to the array under conditions of highstringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS.After hybridization, the array is washed at 55° C. three times asfollows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2%SDS; and 3) third wash in 0.1×SSC.

The arrays are then scanned for green and red fluorescence using aMolecular Dynamics Generation III dual color laser-scanner/detector. Theimages are processed using BioDiscovery Autogene software, and the datafrom each scan set normalized. The experiment is repeated, this timelabeling the two probes with the opposite color in order to perform theassay in both “color directions.” Each experiment is sometimes repeatedwith two more slides (one in each color direction). The data from eachscan is normalized, and the level of fluorescence for each sequence onthe array expressed as a ratio of the geometric mean of 8 replicatespots/genes from the four arrays or 4 replicate spots/gene from 2 arraysor some other permutation.

Normalization: The objective of normalization is to generate a cDNAlibrary in which all transcripts expressed in a particular cell type ortissue are equally represented (S. M. Weissman, Mol. Biol. Med.4(3):133-143 (1987); Patanjali, et al., Proc. Natl. Acad. Sci. USA88(5):1943-1947 (1991)), and therefore isolation of as few as 30,000recombinant clones in an optimally normalized library may represent theentire gene expression repertoire of a cell, estimated to number 10,000per cell.

Total RNA is extracted from harvested cells using RNeasy™ Protect Kit(Qiagen, Valencia, Calif.), following manufacturer's recommendedprocedures. RNA is quantified using RiboGreen™ RNA quantification kit(Molecular Probes, Inc. Eugene, Oreg.). One μg of total RNA is reversetranscribed and PCR amplified using SMART™ PCR cDNA synthesis kit(ClonTech, Palo Alto, Calif.). The cDNA products are size-selected byagarose gel electrophoresis using standard procedures (Sambrook, J. T.,et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, NY). The cDNA is extracted using Bio 101Geneclean® II kit (Qbiogene, Carlsbad, Calif.). Normalization of thecDNA is carried out using kinetics of hybridization principles: 1.0 μgof cDNA is denatured by heat at 100° C. for 10 minutes, then incubatedat 42° C. for 42 hours in the presence of 120 mM NaCl, 10 mM Tris.HCl(pH=8.0), 5 mM EDTA.Na+ and 50% formamide. Single-stranded cDNA(“normalized”) is purified by hydroxyapatite chromatography (#130-0520,BioRad, Hercules, Calif.) following the manufacturer's recommendedprocedures, amplified and converted to double-stranded cDNA by threecycles of PCR amplification, and cloned into plasmid vectors usingstandard procedures (Sambrook, J. T., et al. Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, NY). Allprimers/adaptors used in the normalization and cloning process areprovided by the manufacturer in the SMART™ PCR cDNA synthesis kit(ClonTech, Palo Alto, Calif.). Supercompetent cells (XL-2 BlueUltracompetent Cells, Stratagene, Calif.) are transfected with thenormalized cDNA libraries, plated on solid media and grown overnight at36° C.

The sequences of 10,000 recombinants per normalized library are analyzedby capillary sequencing using the ABI PRISM 3700 DNA Analyzer (AppliedBiosystems, California). To determine the representation of transcriptsin a library, BLAST analysis is performed on the clone sequences toassign transcript identity to each isolated clone, i.e., the sequencesof the isolated polynucleotides are first masked to eliminate lowcomplexity sequences using the XBLAST masking program (Claverie“Effective Large-Scale Sequence Similarity Searches,” Computer Methodsfor Macromolecular Sequence Analysis, Doolittle, ed., Meth. Enzymol.266:212-227 Academic Press, NY, N.Y. (1996); see particularly Claverie,in “Automated DNA Sequencing and Analysis Techniques” Adams et al.,eds., Chap. 36, p. 267 Academic Press, San Diego, 1994 and Claverie etal. Comput. Chem. (1993) 17:191). Generally, masking does not influencethe final search results, except to eliminate sequences of relativelittle interest due to their low complexity, and to eliminate multiple“hits” based on similarity to repetitive regions common to multiplesequences, e.g., Alu repeats. The remaining sequences are then used in aBLASTN vs. GenBank search. The sequences are also used as query sequencein a BLASTX vs. NRP (non-redundant proteins) database search.

Automated sequencing reactions are performed using a Perkin-Elmer PRISMDye Terminator Cycle Sequencing Ready Reaction Kit containing AmpliTaqDNA Polymerase, FS, according to the manufacturer's directions. Thereactions are cycled on a GeneAmp PCR System 9600 as per manufacturer'sinstructions, except that they are annealed at 20° C. or 30° C. for oneminute. Sequencing reactions are ethanol precipitated, pellets areresuspended in 8 microliters of loading buffer, 1.5 microliters isloaded on a sequencing gel, and the data is collected by an ABI PRISM3700 DNA Sequencer. (Applied Biosystems, Foster City, Calif.).

The number of times a sequence is represented in a library is determinedby performing sequence identity analysis on the cloned cDNA sequencesand assigning transcript identity to each isolated clone. First, eachsequence is checked to determine if it is a bacterial, ribosomal, ormitochondrial contaminant. Such sequences are excluded from thesubsequent analysis. Second, sequence artifacts, such as vector andrepetitive elements, are masked and/or removed from each sequence.

The remaining sequences are compared via BLAST (Altschul et al, J. Mol.Biol., 215:40, 1990) to GenBank and EST databases for geneidentification and are compared with each other via FastA (Pearson &Lipman, PNAS, 85:2444, 1988) to calculate the frequency of cDNAappearance in the normalized cDNA library. The sequences are alsosearched against the GenBank and GeneSeq nucleotide databases using theBLASTN program (BLASTN 1.3 MP: Altschul et al., J. Mol. Bio. 215:403,1990). Fourth, the sequences are analyzed against a non-redundantprotein (NRP) database with the BLASTX program (BLASTX 1.3 MP: Altschulet al., supra). This protein database is a combination of theSwiss-Prot, PIR, and NCBI GenPept protein databases. The BLASTX programis run using the default BLOSUM-62 substitution matrix with the filterparameter: “xnu+seg”. The score cutoff utilized is 75. Assembly ofoverlapping clones into contigs is done using the program Sequencher(Gene Codes Corp.; Ann Arbor, Mich.). The assembled contigs are analyzedusing the programs in the GCG package (Genetic Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711) SuiteVersion 10.1.

Example 4 Detection of CA-Sequences in Human Cancer Cells and Tissues

DNA from prostate and breast cancer tissues and other human cancertissues, human colon, normal human tissues including non-cancerousprostate, and from other human cell lines are extracted following theprocedure of Delli Bovi et al. (1986, Cancer Res. 46:6333-6338). The DNAis resuspended in a solution containing 0.05 M Tris HCl buffer, pH 7.8,and 0.1 mM EDTA, and the amount of DNA recovered is determined bymicrofluorometry using Hoechst 33258 dye. Cesarone, C. et al., AnalBiochem 100:188-197 (1979).

Polymerase chain reaction (PCR) is performed using Taq polymerasefollowing the conditions recommended by the manufacturer (Perkin ElmerCetus) with regard to buffer, Mg²⁺, and nucleotide concentrations.Thermocycling is performed in a DNA cycler by denaturation at 94° C. for3 min. followed by either 35 or 50 cycles of 94° C. for 1.5 min., 50° C.for 2 min. and 72° C. for 3 min. The ability of the PCR to amplify theselected regions of the CA gene is tested by using a cloned CApolynucleotide(s) as a positive template(s). Optimal Mg²⁺, primerconcentrations and requirements for the different cycling temperaturesare determined with these templates. The master mix recommended by themanufacturer is used. To detect possible contamination of the master mixcomponents, reactions without template are routinely tested.

Southern blotting and hybridization are performed as described bySouthern, E. M., (J. Mol. Biol. 98:503-517, 1975), using the clonedsequences labeled by the random primer procedure (Feinberg, A. P., etal., 1983, Anal. Biochem. 132:6-13). Prehybridization and hybridizationare performed in a solution containing 6×SSPE, 5% Denhardt's, 0.5% SDS,50% formamide, 100 μg/ml denaturated salmon testis DNA, incubated for 18hrs at 42° C., followed by washings with 2×SSC and 0.5% SDS at roomtemperature and at 37° C. and finally in 0.1×SSC with 0.5% SDS at 68° C.for 30 min (Sambrook et al., 1989, in “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Lab. Press). For paraffin-embedded tissuesections the conditions described by Wright and Manos (1990, in “PCRProtocols”, Innis et al., eds., Academic Press, pp. 153-158) arefollowed using primers designed to detect a 250 bp sequence.

Example 5 Expression of Cloned Polynucleotides in Host Cells

To study the protein products of CA genes, restriction fragments from CADNA are cloned into the expression vector pMT2 (Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress pp 16.17-16.22 (1989)) and transfected into COS cells grown inDMEM supplemented with 10% FCS. Transfections are performed employingcalcium phosphate techniques (Sambrook, et al (1989) pp. 16.32-16.40,supra) and cell lysates are prepared forty-eight hours aftertransfection from both transfected and untransfected COS cells. Lysatesare subjected to analysis by immunoblotting using anti-peptide antibody.

In immunoblotting experiments, preparation of cell lysates andelectrophoresis are performed according to standard procedures. Proteinconcentration is determined using BioRad protein assay solutions. Aftersemi-dry electrophoretic transfer to nitrocellulose, the membranes areblocked in 500 mM NaCl, 20 mM Tris, pH 7.5, 0.05% Tween-20 (TTBS) with5% dry milk. After washing in TTBS and incubation with secondaryantibodies (Amersham), enhanced chemiluminescence (ECL) protocols(Amersham) are performed as described by the manufacturer to facilitatedetection.

Example 6 Generation of Antibodies Against Polypeptides

Polypeptides, unique to CA genes are synthesized or isolated frombacterial or other (e.g., yeast, baculovirus) expression systems andconjugated to rabbit serum albumin (RSA) with m-maleimido benzoic acidN-hydroxysuccinimide ester (MBS) (Pierce, Rockford, Ill.). Immunizationprotocols with these peptides are performed according to standardmethods. Initially, a pre-bleed of the rabbits is performed prior toimmunization. The first immunization includes Freund's complete adjuvantand 500 μg conjugated peptide or 100 μg purified peptide. All subsequentimmunizations, performed four weeks after the previous injection,include Freund's incomplete adjuvant with the same amount of protein.Bleeds are conducted seven to ten days after the immunizations.

For affinity purification of the antibodies, the corresponding CApolypeptide is conjugated to RSA with MBS, and coupled to CNBr-activatedSepharose (Pharmacia, Uppsala, Sweden). Antiserum is diluted 10-fold in10 mM Tris-HCl, pH 7.5, and incubated overnight with the affinitymatrix. After washing, bound antibodies are eluted from the resin with100 mM glycine, pH 2.5.

Example 7 Generation of Monoclonal Antibodies Against a CA Polypeptide

A non-denaturing adjuvant (Ribi, R730, Corixa, Hamilton Mont.) isrehydrated to 4 ml in phosphate buffered saline. 100 μg of thisrehydrated adjuvant is then diluted with 400 μg of Hank's Balanced SaltSolution and this is then gently mixed with the cell pellet used forimmunization. Approximately 500 μg conjugated peptide or 100 μg purifiedpeptide and Freund's complete are injected into Balb/c mice viafoot-pad, once a week. After 6 weeks of weekly injection, a drop ofblood is drawn from the tail of each immunized animal to test the titerof antibodies against CA polypeptides using FACS analysis. When thetiter reaches at least 1:2000, the mice are sacrificed in a CO₂ chamberfollowed by cervical dislocation. Lymph nodes are harvested forhybridoma preparation. Lymphocytes from mice with the highest titer arefused with the mouse myeloma line X63-Ag8.653 using 35% polyethyleneglycol 4000. On day 10 following the fusion, the hybridoma supernatantsare screened for the presence of CAP-specific monoclonal antibodies byfluorescence activated cell sorting (FACS). Conditioned medium from eachhybridoma is incubated for 30 minutes with a combined aliquot of PC3,Colo-205, LnCap, or Panc-1 cells. After incubation, the cell samples arewashed, resuspended in 0.1 ml diluent and incubated with 1 μg/ml of FITCconjugated F(ab′)2 fragment of goat anti-mouse IgG for 30 min at 4° C.The cells are washed, resuspended in 0.5 ml FACS diluent and analyzedusing a FACScan cell analyzer (Becton Dickinson; San Jose, Calif.).Hybridoma clones are selected for further expansion, cloning, andcharacterization based on their binding to the surface of one or more ofcell lines which express the CA polypeptide as assessed by FACS. Ahybridoma making a monoclonal antibody designated mAbCA which binds anantigen designated Ag-CA.x and an epitope on that antigen designatedAg-CA.x.1 is selected.

Example 8 ELISA Assay for Detecting CA Related Antigens

To test blood samples for antibodies that bind specifically torecombinantly produced CA antigens, the following procedure is employed.After a recombinant CA related protein is purified, the recombinantprotein is diluted in PBS to a concentration of 5 μg/ml (500 ng/100 μl).100 microliters of the diluted antigen solution is added to each well ofa 96-well Immulon 1 plate (Dynatech Laboratories, Chantilly, Va.), andthe plate is then incubated for 1 hour at room temperature, or overnightat 4° C., and washed 3 times with 0.05% Tween 20 in PBS. Blocking toreduce nonspecific binding of antibodies is accomplished by adding toeach well 200 μl of a 1% solution of bovine serum albumin in PBS/Tween20 and incubation for 1 hour. After aspiration of the blocking solution,100 μl of the primary antibody solution (anticoagulated whole blood,plasma, or serum), diluted in the range of 1/16 to 1/2048 in blockingsolution, is added and incubated for 1 hour at room temperature orovernight at 4° C. The wells are then washed 3 times, and 100 μl of goatanti-human IgG antibody conjugated to horseradish peroxidase (OrganonTeknika, Durham, N.C.), diluted 1/500 or 1/1000 in PBS/Tween 20, 100 μlof o-phenylenediamine dihydrochloride (OPD, Sigma) solution is added toeach well and incubated for 5-15 minutes. The OPD solution is preparedby dissolving a 5 mg OPD tablet in 50 ml 1% methanol in H₂O and adding50 μl 30% H₂O₂ immediately before use. The reaction is stopped by adding25 l of 4M H₂SO₄. Absorbances are read at 490 nm in a microplate reader(Bio-Rad).

Example 9 Identification and Characterization of CA Antigen on CancerCell Surface

A cell pellet of proximately 25 ul packed cell volume of a cancer cellpreparation is lysed by first diluting the cells to 0.5 ml in waterfollowed by freezing and thawing three times. The solution iscentrifuged at 14,000 rpm. The resulting pellet, containing the cellmembrane fragments, is resuspended in 50 μl of SDS sample buffer(Invitrogen, Carlsbad, Calif.). The sample is heated at 80° C. for 5minutes and then centrifuged for 2 minutes at 14,000 rpm to remove anyinsoluble materials.

The samples are analyzed by Western blot using a 4 to 20% polyacrylamidegradient gel in Tris-Glycine SDS (Invitrogen; Carlsbad Calif.) followingthe manufacturer's directions. Ten microliters of membrane sample areapplied to one lane on the polyacrylamide gel. A separate 10 μL sampleis reduced first by the addition of 2 μL of dithiothreitol (100 mM) withheating at 80° C. for 2 minutes and then loaded into another lane.Pre-stained molecular weight markers SeeBlue Plus2 (Invitrogen;Carlsbad, Calif.) are used to assess molecular weight on the gel. Thegel proteins are transferred to a nitrocellulose membrane using atransfer buffer of 14.4 g/l glycine, 3 g/l of Tris Base, 10% methanol,and 0.05% SDS. The membranes are blocked, probed with a CAP-specificmonoclonal antibody (at a concentration of 0.5 ug/ml), and developedusing the Invitrogen WesternBreeze Chromogenic Kit-AntiMouse accordingto the manufacturer's directions. In the reduced sample of the tumorcell membrane samples, a prominent band is observed migrating at amolecular weight within about 10% of the predicted molecular weight ofthe corresponding CA protein.

Example 10 Preparation of Vaccines

The present invention also relates to a method of stimulating an immuneresponse against cells that express CA polypeptides in a patient usingCA polypeptides of the invention that act as an antigen produced by orassociated with a malignant cell. This aspect of the invention providesa method of stimulating an immune response in a human against cancercells or cells that express CA polynucleotides and polypeptides. Themethod comprises the step of administering to a human an immunogenicamount of a polypeptide comprising: (a) the amino acid sequence of ahuma CA protein or (b) a mutein or variant of a polypeptide comprisingthe amino acid sequence of a human endogenous retrovirus CA protein.

Example 11 Generation of Transgenic Animals Expressing Polypeptides as aMeans for Testing Therapeutics

CA nucleic acids are used to generate genetically modified non-humananimals, or site specific gene modifications thereof, in cell lines, forthe study of function or regulation of prostate tumor-related genes, orto create animal models of diseases, including prostate cancer. The term“transgenic” is intended to encompass genetically modified animalshaving an exogenous CA gene(s) that is stably transmitted in the hostcells where the gene(s) may be altered in sequence to produce a modifiedprotein, or having an exogenous CA LTR promoter operably linked to areporter gene. Transgenic animals may be made through a nucleic acidconstruct randomly integrated into the genome. Vectors for stableintegration include plasmids, retroviruses and other animal viruses,YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs,goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.

The modified cells or animals are useful in the study of CA genefunction and regulation. For example, a series of small deletions and/orsubstitutions may be made in the CA genes to determine the role ofdifferent genes in tumorigenesis. Specific constructs of interestinclude, but are not limited to, antisense constructs to block CA geneexpression, expression of dominant negative CA gene mutations, andover-expression of a CA gene. Expression of a CA gene or variantsthereof in cells or tissues where it is not normally expressed or atabnormal times of development is provided. In addition, by providingexpression of proteins derived from CA in cells in which it is otherwisenot normally produced, changes in cellular behavior can be induced.

DNA constructs for random integration need not include regions ofhomology to mediate recombination. Conveniently, markers for positiveand negative selection are included. For various techniques fortransfecting mammalian cells, see Keown et al., Methods in Enzymology185:527-537 (1990).

For embryonic stem (ES) cells, an ES cell line is employed, or embryoniccells are obtained freshly from a host, e.g. mouse, rat, guinea pig,etc. Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of appropriate growth factors, such as leukemiainhibiting factor (LIF). When ES cells are transformed, they may be usedto produce transgenic animals. After transformation, the cells areplated onto a feeder layer in an appropriate medium. Cells containingthe construct may be detected by employing a selective medium. Aftersufficient time for colonies to grow, they are picked and analyzed forthe occurrence of integration of the construct. Those colonies that arepositive may then be used for embryo manipulation and blastocystinjection. Blastocysts are obtained from 4 to 6 week old superovulatedfemales. The ES cells are trypsinized, and the modified cells areinjected into the blastocoel of the blastocyst. After injection, theblastocysts are returned to each uterine horn of pseudopregnant females.Females are then allowed to go to term and the resulting chimericanimals screened for cells bearing the construct. By providing for adifferent phenotype of the blastocyst and the ES cells, chimeric progenycan be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs are maintained as allogeneic orcongenic grafts or transplants, or in in vitro culture. The transgenicanimals may be any non-human mammal, such as laboratory animals,domestic animals, etc. The transgenic animals are used in functionalstudies, drug screening, etc., e.g. to determine the effect of acandidate drug on prostate cancer, to test potential therapeutics ortreatment regimens, etc.

Example 12 Diagnostic Imaging Using CA Specific Antibodies

The present invention encompasses the use of antibodies to CApolypeptides to accurately stage cancer patients at initial presentationand for early detection of metastatic spread of cancer.Radioimmunoscintigraphy using monoclonal antibodies specific for CApolypeptides can provide an additional cancer-specific diagnostic test.The monoclonal antibodies of the instant invention are used forhistopathological diagnosis of carcinomas.

Subcutaneous human xenografts of cancer cells in nude mice is used totest whether a technetium-99m (^(99m)Tc)-labeled monoclonal antibody ofthe invention can successfully image the xenografted cancer by externalgamma scintography as described for seminoma cells by Marks, et al.,Brit. J. Urol. 75:225 (1995). Each monoclonal antibody specific for a CApolypeptide is purified from ascitic fluid of BALB/c mice bearinghybridoma tumors by affinity chromatography on protein A-Sepharose.Purified antibodies, including control monoclonal antibodies such as anavidin-specific monoclonal antibody (Skea, et al., J. Immunol. 151:3557(1993)) are labeled with ^(99m)Tc following reduction, using the methodsof Mather, et al., J. Nucl. Med. 31:692 (1990) and Zhang et al., Nucl.Med. Biol. 19:607 (1992). Nude mice bearing human cancer cells areinjected intraperitoneally with 200-500 μCi of ^(99m)Tc-labeledantibody. Twenty-four hours after injection, images of the mice areobtained using a Siemens ZLC3700 gamma camera equipped with a 6 mmpinhole collimator set approximately 8 cm from the animal. To determinemonoclonal antibody biodistribution following imaging, the normal organsand tumors are removed, weighed, and the radioactivity of the tissuesand a sample of the injectate are measured. Additionally, CA-specificantibodies conjugated to antitumor compounds are used forcancer-specific chemotherapy.

Example 13 Immunohistochemical Methods

Frozen tissue samples from cancer patients are embedded in an optimumcutting temperature (OCT) compound and quick-frozen in isopentane withdry ice. Cryosections are cut with a Leica 3050 CM mictrotome atthickness of 5 μm and thaw-mounted on vectabound-coated slides. Thesections are fixed with ethanol at −20° C. and allowed to air dryovernight at room temperature. The fixed sections are stored at −80° C.until use. For immunohistochemistry, the tissue sections are retrievedand first incubated in blocking buffer (PBS, 5% normal goat serum, 0.1%Tween 20) for 30 minutes at room temperature, and then incubated withthe CA protein-specific monoclonal antibody and control monoclonalantibodies diluted in blocking buffer (1 μg/ml) for 120 minutes. Thesections are then washed three times with the blocking buffer. The boundmonoclonal antibodies are detected with a goat anti-mouse IgG+IgM (H+L)F(ab′)²-peroxidase conjugates and the peroxidase substratediaminobenzidine (1 mg/ml, Sigma Catalog No. D 5637) in 0.1 M sodiumacetate buffer pH 5.05 and 0.003% hydrogen peroxide (Sigma cat. No.H1009). The stained slides are counter-stained with hematoxylin andexamined under Nikon microscope.

Monoclonal antibody against a CA protein (antigen) is used to testreactivity with various cell lines from different types of tissues.Cells from different established cell lines are removed from the growthsurface without using proteases, packed and embedded in OCT compound.The cells are frozen and sectioned, then stained using a standard IHCprotocol. The CellArray™ technology is described in WO 01/43869. Normaltissue (human) obtained by surgical resection are frozen and mounted.Cryosections are cut with a Leica 3050 CM mictrotome at thickness of 5μm and thaw-mounted on vectabound-coated slides. The sections are fixedwith ethanol at −20° C. and allowed to air dry overnight at roomtemperature. PolyMICA™ Detection kit is used to determine binding of aCA-specific monoclonal antibody to normal tissue. Primary monoclonalantibody is used at a final concentration of 1 μg/ml.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1-67. (canceled)
 68. A method of diagnosing cancer in a patientcomprising detecting the presence of differential expression of a genein a patient sample, said gene comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44, 50, 60,82, 88, 112, 122, 128, 136, 154, and 170, wherein the presence ofdifferential expression of the gene in said sample is indicative of apatient who has cancer.
 69. The method of claim 68 wherein the level ofgene expression is determined by measuring mRNA levels, said mRNA havinga sequence selected from the group consisting of SEQ ID NO: 5, 11, 13,19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65, 67, 69, 71, 73, 75,83, 89,91,93,95,97,99, 101, 103, 105, 107, 113, 115, 117, 123, 129, 137,139, 141, 143, 145, 147, 149, 155, 157, 159, 161, 163, 165, 171, and173, or the complement thereof.
 70. The method of claim 68 wherein thedifferential expression is upregulation of expression of the gene ascompared to a control.
 71. A method of screening for anti-canceractivity comprising: (a) contacting a cell that expresses a gene with acandidate anti-cancer agent, said gene comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44,50, 60, 82, 88, 112, 122, 128, 136, 154, and 170; and (b) detecting adifference between the level of gene expression in the cell in thepresence and in the absence of the candidate anti-cancer agent, whereina difference between the level of gene expression in the cell in thepresence and in the absence of the candidate anti-cancer agent indicatesthat the candidate anti-cancer agent has anti-cancer activity.
 72. Themethod of claim 71 wherein the candidate anti-cancer agent is anantibody, small organic compound, small inorganic compound, orpolynucleotide.
 73. The method of claim 71 wherein the candidateanti-cancer agent is a monoclonal antibody.
 74. The method of claim 72wherein the antibody is a human antibody or humanized antibody.
 75. Themethod of claim 71 wherein the polynucleotide is an antisenseoligonucleotide or an siRNA.
 76. A method of screening drug candidatescomprising: a) providing a cell that expresses a gene comprising anucleic acid sequence selected from the group consisting of SEQ ID NO:4, 10, 18, 26, 34, 44, 50, 60, 82, 88, 112, 122, 128, 136, 154, and 170,or fragment thereof; b) adding a drug candidate to said cell; and c)determining the effect of said drug candidate on the expression of saidgene.
 77. The method of claim 76 wherein said gene encodes an mRNAhaving a sequence selected from the group consisting of SEQ ID NO: 5,11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63, 65, 67, 69, 71,73, 75, 83, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 113, 115, 117,123, 129, 137, 139, 141, 143, 145, 147, 149, 155, 157, 159, 161, 163,165, 171, and 173, or the complement thereof.
 78. The method of claim 76wherein said determining comprises comparing the level of expression ofthe gene in the absence of said drug candidate to the level ofexpression in the presence of said drug candidate.
 79. A method ofscreening for a bioactive agent capable of binding to a protein, whereinsaid protein is encoded by a nucleic acid comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 4, 10, 18, 26,34, 44, 50, 60, 82, 88, 112, 122, 128, 136, 154, and 170, said methodcomprising: a) combining said protein and a candidate bioactive agent;and b) determining the binding of said candidate agent to said protein.80. A method for screening for a bioactive agent capable of modulatingthe activity of a protein, wherein said protein is encoded by a nucleicacid comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 4, 10, 18, 26, 34, 44, 50, 60, 82, 88, 112,122, 128, 136, 154, and 170, said method comprising: a) combining saidprotein and a candidate bioactive agent; and b) determining the effectof said candidate agent on the bioactivity of said protein.
 81. Themethod of claim 80 wherein the protein has a sequence selected from thegroup consisting of SEQ ID NO: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46,52, 54, 56, 62, 64, 66, 68, 70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 114, 116, 118, 124, 130, 138, 140, 142, 144, 146,148, 150, 156, 158, 160, 162, 164, 166, 172, and
 174. 82. A method ofevaluating the effect of a candidate cancer drug comprising: a)administering the drug to a patient; b) removing a cell sample from saidpatient; and c) determining alterations in the expression or activationof a gene in the cell sample comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44, 50, 60,82, 88, 112, 122, 128, 136, 154, and
 170. 83. The method of claim 82wherein gene expression is determined by measuring mRNA levels of saidone or more genes, said mRNA having a sequence selected from the groupconsisting of SEQ ID NO: 5, 11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53,55, 61, 63, 65, 67, 69, 71, 73, 75, 83, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 113, 115, 117, 123, 129, 137, 139, 141, 143, 145, 147,149, 155, 157, 159, 161, 163, 165, 171, and 173, or the complementthereof.
 84. A method of diagnosing cancer comprising: a) determiningthe expression of one or more genes comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44,50, 60, 82, 88, 112, 122, 128, 136, 154, and 170, in a first tissue typeof a first individual; and b) comparing the gene expression in a) toexpression of the gene in a control, wherein a difference between theexpression of the gene in the first tissue type of the first individualcompared to expression of the gene in the control indicates that thefirst individual has cancer.
 85. The method of claim 84 wherein thecontrol is a known normal sample.
 86. A method for inhibiting theactivity of a protein, wherein said protein is encoded by a nucleic acidcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 5, 11, 13, 19, 27, 29, 35, 37, 39, 45, 51, 53, 55, 61, 63,65, 67, 69, 71, 73, 75, 83, 89, 91, 93,95,97,99, 101, 103, 105, 107,113, 115, 117, 123, 129, 137, 139, 141, 143, 145, 147, 149, 155, 157,159, 161, 163, 165, 171, and 173, said method comprising binding aninhibitor to said protein.
 87. The method of claim 86 wherein theprotein has a sequence selected from the group consisting of SEQ ID NO:6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68, 70,72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114, 116,118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160, 162,164, 166, 172, and
 174. 88. A method of treating cancer comprisingadministering to a patient an inhibitor of a protein, wherein saidprotein is encoded by a nucleic acid comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44,50, 60, 82, 88, 112, 122, 128, 136, 154, and
 170. 89. The method ofclaim 88 wherein the protein has a sequence selected from the groupconsisting of SEQ ID NO: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54,56, 62, 64, 66, 68, 70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 114, 116, 118, 124, 130, 138, 140, 142, 144, 146, 148,150, 156, 158, 160, 162, 164, 166, 172, and
 174. 90. A method ofneutralizing the effect of a protein, wherein said protein is encoded bya nucleic acid comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 4, 10, 18, 26, 34, 44, 50, 60, 82, 88,112, 122, 128, 136, 154, and 170, the method comprising contacting anagent specific for said protein with said protein in an amountsufficient to effect neutralization.
 91. The method of claim 90 whereinthe protein has a sequence selected from the group consisting of SEQ IDNO: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68,70, 72, 74, 76, 84, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 114,116, 118, 124, 130, 138, 140, 142, 144, 146, 148, 150, 156, 158, 160,162, 164, 166, 172, and
 174. 92. An isolated antibody which specificallybinds to a polypeptide having a sequence selected from the groupconsisting of a sequence selected from the group consisting of SEQ IDNO: 6, 12, 14, 20, 28, 30, 36, 38, 40, 46, 52, 54, 56, 62, 64, 66, 68,70, 72, 74, 76, 84, 90, 92, 94,96,98,100,102,104,106,108,114,116,118,124,130,138,140,142,144,146,148,150,156,158, 160, 162, 164, 166, 172, and
 174. 93. The isolated antibody ofclaim 92 wherein the antibody is a monoclonal antibody
 94. The isolatedantibody of claim 92 wherein the antibody is a human or humanizedantibody.